Human islet amyloid polypeptide (hiapp) specific antibodies and uses thereof

ABSTRACT

Provided are novel human islet amyloid polypeptide, also known as amylin and IAPP and proIAPP respectively, specific antibodies as well as fragments, derivatives and variants thereof as well as methods related thereto. Assays, kits, and solid supports related to antibodies specific for IAPP and/or proIAPP are also disclosed. The antibody, immunoglobulin chain(s), as well as binding fragments, derivatives and variants thereof can be used in pharmaceutical and diagnostic compositions for IAPP and/or proIAPP targeted immunotherapy and diagnostics, respectively.

FIELD OF THE INVENTION

The present invention generally relates to novel molecules specificallybinding to human islet amyloid polypeptide (hIAPP) also known as amylinand/or to its precursor proislet amyloid polypeptide (proIAPP),particularly human antibodies as well as fragments, derivatives andvariants thereof that recognize the IAPP, proIAPP proteins, aggregatedforms of IAPP, aggregated forms of proIAPP, and/or IAPP fibrils. Inaddition, the present invention relates to pharmaceutical and diagnosticcompositions comprising such binding molecules, antibodies and mimicsthereof valuable both as a diagnostic tool to identify IAPP, proIAPP,aggregated IAPP, proIAPP species and/or IAPP fibrils in plasma and alsoin passive vaccination strategies for treating disorders related toaggregated IAPP, aggregated proIAPP, and IAPP fibrils such as diabetesmellitus type 2 (T2D) and islet rejection following clinical pancreaticislet transplantation into individuals with diabetes mellitus type 1(T1D).

BACKGROUND OF THE INVENTION

Protein accumulation, modifications and aggregation are pathologicalaspects of numerous metabolic diseases including well knownneurodegenerative diseases such as Huntington's, Alzheimer's (AD) andParkinson's diseases (PD) (Taylor et al., Science 296 (2005),1991-1995). Pathological protein aggregation is also involved inmetabolic diseases such as diabetes mellitus type 2 (T2D) and isletrejection following clinical pancreatic islet transplantation intoindividuals with diabetes mellitus type 1 (T1D). Misfolding andaggregation of proteins lead to the development of amyloid deposits andseem to be directly related to cell toxicity in these diseases. Isletamyloid polypeptide (IAPP or amylin), a physiological peptideco-secreted with insulin by β-cells in the pancreas, forms fibrillaraggregates in pancreatic islets (also called islets of Langerhans) ofT2D patients and has been suggested to play a role in the development ofthe disease (Westermark et al. (2011), Physiol. Rev. 91(3): 795-826).Furthermore, as mentioned before, IAPP aggregates have been found inpancreatic islets upon transplantation of isolated islets in patientswith diabetes mellitus type 1 (T1D).

Human IAPP (hIAPP) is a peptide hormone that consists of 37 amino acids,with a disulfide bridge between cysteine residues 2 and 7 and anamidated C-terminus. Pancreatic islets are composed of 65 to 80%β-cells, which produce and secrete insulin and IAPP essential forregulation of blood glucose levels and cell metabolism. IAPP isprocessed from preprohormone preproIAPP, a 89 amino acid precursorproduced in pancreatic β-cells.

PreproIAPP is rapidly cleaved after translation into proislet amyloidpolypepide, a 67 amino acid peptide, which undergoes additionalproteolysis and post-translational modifications to generate hIAPP.hIAPP expression is regulated together with insulin, as increasedinsulin production leads to increased hIAPP levels. hIAPP is releasedfrom pancreatic β-cells into the blood circulation and is involved inglycemic regulation through gastric emptying and satiety control, insynergy with insulin.

While hIAPP acts as a regulator of cell metabolism under physiologicalconditions, hIAPP can aggregate and form amyloid fibrils (IAPPamyloidosis) associated with β-cell failure, increased β-cell death andreduced β-cell mass. Several evidences point toward hIAPP amyloidosis asa major trigger for T2D pathogenesis. First, deposition of hIAPP fibrilsis found in more than 90% of type-2 diabetes patients (Zraika et al.(2010), Diabetologia 53(6): 1046-1056). Second, hIAPP aggregation istoxic to β-cells and correlates with the reduction in insulin producingβ-cells (Butler et al. (2003), Diabetes 52(9): 2304-2314; Ritzel et al.(2007), Diabetes 56(1): 65-71; Jurgens et al. (2011), Am. J. Pathol.178(6): 2632-2640). Third, transgenic murine models expressing hIAPPshow pancreatic islet amyloid deposits and spontaneously develop T2D(Janson et al. (1996), Proc. Natl. Acad. Sci. USA 93(14): 7283-7288;Hoppener et al. (1999), Diabetologia 42(4): 427-434; Hull et al. (2003),Diabetes 52(2): 372-379; Butler et al. (2004), Diabetes 53(6):1509-1516; Matveyenko et al. (2006), ILAR J. 47(3): 225-233; Hoppener etal. (2008), Exp. Diabetes Res. 697035). They recapitulate the humandisease with β-cell dysfunction, β-cell mass deficiency and β-cell loss,comparable to what observed in the tissues from T2D patients. hIAPPexpression and amyloid formation directly correlate with β-cellapoptosis and diabetes development in these models, thus providingevidence for the contribution of human IAPP in the development of thedisease. Moreover, treatment interfering with hIAPP aggregationameliorated the diabetic phenotype and increased animal life span(Aitken et al. (2009), Diabetes 59(1): 161-171). hIAPP aggregation andamyloidosis is a prerequisite for toxicity. The non-amyloidogenic rodentIAPP (rIAPP), which is unable to form fibrils as a result of six aminoacid substitution, is nontoxic to β-cells. In the development of thedisease, pathological hIAPP aggregation found in human pancreatic isletsmay cause β-cell dysfunction and death associated with impairment ofinsulin secretion. In addition, compensatory increase in β-cell mass andinsulin and amylin secretion to maintain normal blood glucose levels mayfavor the formation of toxic hIAPP oligomers and deposition of hIAPPfibrils. While initial hIAPP oligomers are considered as the maincytotoxic species, the hIAPP fibril end product may also play a role inβ-cell loss (Meier et al. (2006), Am. J. Physiol. Endocrinol. Metab.291(6): E1317-1324; Haataja et al. (2008), Endocr. Rev. 29(3): 303-316;Engel et al. (2008), Proc. Natl. Acad. Sci. USA 105(16): 6033-6038).hIAPP fibrils have also been observed in isolated pancreatic islets fromdonors and associated to β-cell loss following clinical pancreaticislets transplantation into individuals with type-1 diabetes (Anderssonet al. (2008), Exp. Diabetes Res. 562985; Udayasankar et al. (2009),Diabetologia 52(1): 145-153; Bohman et al. (2012), Amyloid 19(2):87-93). The exact mechanism leading to hIAPP aggregation and amyloidosisin T2D is unknown. Insulin resistance in T2D increases insulin secretiondemand together with proIAPP cell content and hIAPP release, what mayelicit amyloidosis as hIAPP fibril formation is concentration dependent.Another proposed mechanism is the accumulation and aggregation ofN-terminal unprocessed proIAPP caused by proteolysis failure in thesetting of insulin resistance, as partially processed forms of proIAPPare found in amyloid deposits, in particular the 48 residue intermediateproIAPP₁₋₄₈ (Marzban et al. (2006), Diabetes 55(8): 2192-2201). In thiscontext, abnormal processing of proIAPP may act as a seed for hIAPPamyloidosis and increase amyloid formation (Paulsson et al. (2005),Diabetes 54(7): 2117-2125; Paulsson et al. (2006), Diabetologia 49(6):1237-1246; Marzban et al. (2006), Diabetes 55(8): 2192-2201). ProIAPP istherefore also considered as an appropriate therapeutic target.

Clinical features of T2D are high blood glucose levels and insulinresistance and/or deficiency. Diabetes mellitus is a group of metabolicdiseases including T1D, T2D, and gestational diabetes. T2D, also namedadult-onset diabetes, obesity-related diabetes, and noninsulin-dependentdiabetes mellitus (NIDDM) is the most common form of diabetes,accounting for about 90% of all cases (Gerich et al. (1998), Endocr.Rev. 19(4): 491-503). T2D is characterized by a decrease in the numberof functional insulin-producing β-cells. While the pathology progresses,it can lead to long-term complications such as cardiovascular disease,diabetic retinopathy leading to blindness, kidney failure, frequentinfections, and amputations caused by poor circulation. As aconsequence, T2D is associated with a shorter life expectancy. Thedisease affects more than 300 million people worldwide resulting in morethan a million deaths annually. Both genetic determinants andenvironmental factors lead to the development of the disease, withobesity, physical inactivity and aging thought to be the primary cause(Kahn et al. (2006), Nature 444(7121): 840-846).

Current treatments for T2D include lifestyle management (diet andexercise) and pharmacological intervention such as metformin and insulinsupply to decrease blood glucose levels by either stimulating thepancreas to release insulin or increasing insulin response. Thesetreatments are based on symptomatic improvement of diabetes, with theconsequence of a lack of durability. Indeed, none of the availabletreatments have been shown to counteract the aggregation of hIAPP andthe loss of pancreatic β-cells. New treatment strategies involvinganalogues of glucagon-peptide 1 (GLP-1) (Butler et al. (2009),Diabetologia 53(1): 1-6) and inhibitors of GLP-1 inactivating enzymedipeptidyl-peptidase 4 (DDP4) are based on the potent insulinotropiceffect of GLP-1 and its effect to enhance β-cell proliferation.Importantly, increased insulin release is also coupled to increasedamylin release. Experimentally, stimulated insulin secretion has beenshown to promote the development of islet amyloidosis in animal modelsand similar effects can be expected in humans (Aston-Mourney et al.(2011), Diabetologia 54(7): 1756-1765). These treatments could thereforepotentially aggravate islet amyloidosis. More recent and promisingstrategies involve the development of anti-inflammatory drugs orantibodies targeting the IL-1β pathway (Donath et al. (2008), Nat. Clin.Pract. Endocrinol. Metab. 4(5): 240-241; Ehes et al. (2009), Proc. Natl.Acad. Sci. USA 106(33): 13998-14003; Owyang et al. (2010), Endocrinology151(6): 2515-2527; Dinarello et al. (2010), Curr. Opin. Endocrinol.Diabetes Obes. 17(4): 314-321; Boni-Schnetzler et al. (2011), J. Clin.Endocrinol. Metab. 93(10): 4065-4074; Boni-Schnetzler et al. (2012), Br.J. Clin. Pharmacol.; Cavelti-Weder et al. (2012), Diabetes Care). Ofimportant note, recent studies show that hIAPP specifically induce theinflammasome—IL-1β system leading to activation of the innate immunesystem (Masters et al. (2010), Nat. Immunol. 11(10): 897-904;Mandrup-Poulsen et al. (2010), Nat. Immunol. 11(10): 881-883), thussupporting a therapeutic strategy targeting hIAPP aggregation.

These findings highlight the potential benefit associated with active orpassive immunotherapy approaches targeting hIAPP and/or proIAPP.

Summarizing the above, novel therapeutic strategies are urgently neededaddressing aggregated hIAPP, proIAPP proteins and/or hIAPP oligomersand/or fibrils with efficacious and safe therapy.

Passive immunization with human antibodies which are evolutionarilyoptimized and affinity matured by the human immune system would providea promising new therapeutic avenue with a high probability for excellentefficacy and safety.

SUMMARY OF THE INVENTION

The present invention makes use of the hIAPP-specific immune response ofhealthy human subjects for the isolation of natural anti-hIAPP specifichuman monoclonal antibodies. In particular, experiments performed inaccordance with the present invention were successful in the isolationof monoclonal hIAPP and/or proIAPP-specific antibodies from a pool ofhealthy human subjects or from pools of obese patients and otherpatients groups with enhanced risk to develop T2D, which at the time ofantibody isolation showed no signs of T2D.

The present invention is thus directed to human antibodies,antigen-binding fragments and similar antigen-binding molecules whichare capable of specifically recognizing IAPP and/or proIAPP. If notindicated otherwise, by “specifically recognizing IAPP and/or proIAPP”,“antibody specific to/for IAPP and/or proIAPP” and “anti-IAPP and/oranti-proIAPP antibody” is meant specifically, generally, andcollectively antibodies to the native monomeric form of IAPP; antibodiesto the proIAPP precursor form of IAPP; antibodies binding specificallyto either forms, IAPP and proIAPP; antibodies binding to aggregated,oligomeric, fibrillar and/or non-fibrillar IAPP and/or proIAPP species.Provided herein are human antibodies selective for full-length, and/oraggregated forms, such as oligomeric, fibrillar and non-fibrillaraggregated forms of IAPP and/or proIAPP.

In a particularly preferred embodiment of the present invention, thehuman antibody or antigen-binding fragment thereof demonstrates theimmunological binding characteristics of an antibody characterized bythe variable regions V_(H) and/or V_(L) as set forth in FIG. 1 or FIG.2.

The antigen-binding fragment of the antibody can be a single chain Fvfragment, an F(ab′) fragment, an F(ab) fragment, and an F(ab′)2fragment, or any other antigen-binding fragment. In a specificembodiment, infra, the antibody or fragment thereof is a human IgGisotype antibody. Alternatively, the antibody is a chimeric human-rodentor rodentized antibody such as murine or murinized, rat or ratinizedantibody, the rodent versions being particularly useful for diagnosticmethods and studies in animals.

Furthermore, the present invention relates to compositions comprisingthe antibody of the present invention or active fragments thereof and toimmunotherapeutic and immunodiagnostic methods using such compositionsin the prevention, diagnosis or treatment of disorders related to IAPP,such as T2D, wherein an effective amount of the composition isadministered to a patient in need thereof.

Naturally, the present invention extends to the immortalized human Bmemory lymphocyte and B cell, respectively, that produces the antibodyor an antigen binding fragment thereof having the distinct and uniquecharacteristics as defined below.

The present invention also relates to polynucleotides encoding at leasta variable region of an immunoglobulin chain of the antibody of theinvention. Preferably, said variable region comprises at least onecomplementarity determining region (CDR) of the V_(H) and/or V_(L) ofthe variable region as set forth in FIG. 1 or in FIG. 2.

Accordingly, the present invention also encompasses vectors comprisingsaid polynucleotides and host cells transformed therewith as well astheir use for the production of an antibody and equivalent bindingmolecules which are specific for IAPP and/or proIAPP. Means and methodsfor the recombinant production of antibodies and mimics thereof as wellas methods of screening for competing binding molecules, which may ormay not be antibodies, are known in the art. However, as describedherein, in particular with respect to therapeutic applications in humanthe antibody of the present invention is a human antibody in the sensethat application of said antibody is substantially free of an immuneresponse directed against such antibody otherwise observed for chimericand even humanized antibodies.

Furthermore, disclosed herein are compositions and methods that can beused to identify IAPP and/or proIAPP in samples and/or in vivo. Thedisclosed anti-IAPP and/or proIAPP antibodies or IAPP and/or proIAPPbinding fragments thereof can be used to screen human blood, plasma,serum, saliva, peritoneal fluid, cerebrospinal fluid (“CSF”), and urinefor the presence of IAPP and/or proIAPP in samples, for example, byusing ELISA-based or surface adapted assay. In one embodiment thepresent invention relates to a method of diagnosing or monitoring theprogression of a disorder related to IAPP and/or proIAPP in a subject,the method comprising determining the presence of IAPP and/or proIAPPoligomers, aggregates or fibrils in a sample from the subject to bediagnosed with at least one antibody of the present invention or an IAPPand/or proIAPP binding molecule having substantially the same bindingspecificities of any one thereof, wherein the presence of IAPP and/orproIAPP oligomers, aggregates or fibrils is indicative of the disorder.

Furthermore, in one embodiment of the present invention the disclosedanti-IAPP and/or proIAPP antibodies or IAPP and/or proIAPP bindingfragments thereof and/or IAPP and/or proIAPP binding moleculescomprising at least one CDR of an antibody of the present invention areprovided for the preparation of a composition for in vivo detection(also called in vivo imaging) of or targeting a therapeutic and/ordiagnostic agent to IAPP and/or proIAPP in the human or animal body. Themethods and compositions disclosed herein can aid in disorders relatedto IAPP and characterized, e.g., by the occurrence of oligomeric,fibrillar and non-fibrillar aggregated forms of IAPP and/or proIAPP suchas T2D diagnosis and can be used to monitor disease progression andtherapeutic efficacy of the therapy provided to the subject, for examplein in vivo imaging related diagnostic methods. Therefore, in oneembodiment the IAPP and/or proIAPP binding molecule of the presentinvention is provided, wherein said in vivo detection (imaging)comprises positron emission tomography (PET), single photon emissiontomography (SPECT), near infrared (NIR) optical imaging or magneticresonance imaging (MRI).

Hence, it is a particular object of the present invention to providemethods for treating, diagnosing or preventing a disease related tofibrillar and/or non-fibrillar oligomeric and/or aggregated, IAPP and/orproIAPP such as Type 2 Diabetes (T2D). The methods compriseadministering an effective concentration of a human antibody or antibodyderivative to the subject where the antibody targets IAPP and/orproIAPP.

In a further aspect the present invention provides a peptide having anepitope of IAPP and/or proIAPP specifically recognized by an antibody ofthe present invention. Said peptide comprises or consists of an aminoacid sequence as indicated below in the detailed description and in theexamples or a modified sequence thereof in which one or more amino acidsare substituted, deleted and/or added. Additionally, the presentinvention provides a method for diagnosing T2D or the risk to developT2D in a subject, comprising a step of determining the presence of anantibody that binds to said peptide in a biological sample of saidsubject. Further embodiments of the present invention will be apparentfrom the description and Examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Amino acid and nucleotide sequences of the variable region, i.e.heavy chain and kappa/lambda light chain of human IAPP antibodiesNI-203.9A2 (A), NI-203.19H8 (B), NI-203.26C11 (C), NI-203.8E3 (D),NI-203.11B12 (E), NI-203.205F8 (F), NI-203.9B3 (G), NI-203.19F2 (H), andNI-203.15C7 (I). Framework (FR) and complementarity determining regions(CDRs) are indicated with the CDRs being underlined. The heavy chainjoining region (JH) and light chain joining region (JK) are indicated aswell. Due to the cloning strategy the amino acid sequence at theN-terminus of the heavy chain and light chain may potentially containprimer-induced alterations in FR1, which however do not substantiallyaffect the biological activity of the antibody. In order to provide aconsensus human antibody, the nucleotide and amino acid sequences of theoriginal clone were aligned with and tuned in accordance with thepertinent human germ line variable region sequences in the database;see, e.g., Vbase (http://vbase.mrc-cpe.cam.ac.uk/) hosted by the MRCCentre for Protein Engineering (Cambridge, UK). The amino acid sequenceof human antibodies is indicated when N-terminus amino acids areconsidered to potentially deviate from the consensus germ line sequencedue to the PCR primer and thus have been replaced by primer-inducedmutation correction (PIMC). PIMC-modified amino acids are indicated inbold on the sequences.

FIG. 2: Amino acid and nucleotide sequences of the variable region, i.e.heavy chain and kappa/lambda light chain of human proIAPP antibodiesNI-203.1D10 (A), NI-203.2A11 (B), NI-203.10C4 (C), NI-203.20H9 (D),NI-203.26D2 (E) and NI-203.60H3 (F). Framework (FR) and complementaritydetermining regions (CDRs) are indicated with the CDRs being underlined.The heavy chain joining region (JH) and light chain joining region (JK)are indicated as well. Due to the cloning strategy the amino acidsequence at the N-terminus of the heavy chain and light chain maypotentially contain primer-induced alterations in FR1, which however donot substantially affect the biological activity of the antibody. Inorder to provide a consensus human antibody, the nucleotide and aminoacid sequences of the original clone were aligned with and tuned inaccordance with the pertinent human germ line variable region sequencesin the database; see, e.g., Vbase (http://vbase.mrc-cpe.cam.ac.uk/)hosted by the MRC Centre for Protein Engineering (Cambridge, UK). Theamino acid sequence of human antibodies is indicated when N-terminusamino acids are considered to potentially deviate from the consensusgerm line sequence due to the PCR primer and thus have been replaced byprimer-induced mutation correction (PIMC). PIMC-modified amino acids areindicated in bold on the sequences.

FIG. 3: IAPP-binding specificity of human recombinant antibodiesassessed by direct ELISA. (A) Electron microscopy image of the IAPPsolution (2 mg/ml) used for ELISA plate coating. Scale bar represents 1(B) Recombinant NI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3showed a specific binding to human IAPP (10 μg/ml). BSA (10 μg/ml) wasused as a control to determine unspecific binding. Data are expressed asOD values at 450 nm.

FIG. 4: EC₅₀ determination of the recombinant human-derived anti-IAPPantibodies for IAPP and proIAPP. (A) Electron microscopy images of theIAPP and proIAPP solutions (2 mg/ml) used for ELISA plate coating. Scalebar represents 1 (B) Plates were incubated with the indicatedconcentrations of recombinant human-derived antibodies NI-203.9A2,NI-203.19H8, NI-203.26C11 or NI-203.8E3. The antibodies NI-203.9A2,NI-203.19H8, NI-203.26C11 and NI-203.8E3 bind with high affinity tohuman IAPP (▪, 10 μg/ml) with an EC₅₀ of 9 nM, 22 nM, 6 nM and 4 nM,respectively. NI-203.26C11 also binds proIAPP (□, 10 μg/ml) with an EC₅₀of 260 nM. Measurements were made in duplicate and background signal onBSA was subtracted. Data are expressed as mean OD values at 450 nm.

FIG. 5: Human-derived anti-IAPP antibodies are specific to IAPP fibrils.(A) Electron microscopy images of IAPP (2 mg/ml) and nonfibrillar IAPP(500 μg/ml) solutions used for ELISA plate coating. While the IAPPsolution contains fibrils, IAPP fibrils are lacking in the nonfibrillarIAPP solution. Scale bar represents 1 (B) Plates were incubated with theindicated concentrations of recombinant human-derived antibodiesNI-203.9A2, NI-203.19H8, NI-203.26C11 or NI-203.8E3. NI-203.9A2,NI-203.19H8, NI-203.26C11 and NI-203.8E3 antibodies bind with highaffinity to IAPP fibrils (IAPP solution, ▪, 10 μg/ml) and with very lowaffinity to nonfibrillar IAPP (Δ, 10 μg/ml), suggesting specificitytoward IAPP fibrils. Measurements were made in duplicate and backgroundsignal on BSA was subtracted. Data are expressed as mean OD values at450 nm.

FIG. 6: IAPP binding epitopes of human recombinant antibodies assessedby pepscan analysis. (A) Pepscan images of recombinant NI-203.19H8 andNI-203.26C11 human-derived antibodies (1 μg/ml). NI-203.19H8 bindingoccurred at peptides 6 and 7 (row A) covering amino acids 19-25 (peptide6: 16-LVHSSNNFGA-25 SEQ ID NO: 6, peptide 7: 19-SSNNFGAILS-28 SEQ ID NO:8, consensus binding sequence: 19-SSNNFGA-25 SEQ ID NO: 4). NI-203.26C11binding occurred at peptide 1 (row A) covering amino acids 1-10 (peptide1: 1-KCNTATCATQ-10 SEQ ID NO: 9) but not at peptide 2 covering aminoacids 4-13 (peptide 2: 4-TATCATQRLA-13 SEQ ID NO: 10). Alaninesubstitution or replacement at residues 2-8 on peptides 33-39 and 41(row C) impaired NI-203.26C11 binding (peptide 33 mutation: C2A, peptide34 mutation: N3A, peptide 35 mutation: T4A, peptide 36 mutation: ASG,peptide 37 mutation: ASP, peptide 38 mutation: T6A, peptide 39 mutation:C7A, peptide 41 mutation: A8P). Secondary HRP-conjugated donkeyanti-human IgG Fcγ only (1:20000; IIary Ab) was used as a control. (B)Identified binding epitopes of the different human-derived IAPP-specificantibodies within the indicated amino acids of the human IAPP proteinsequence. Upper panel: amino acid sequence of the full-length human IAPP(amino acids 1-37). NI: binding epitope not identified.

FIG. 7: NI-203.9A2, NI-203.19H8 and NI-203.26C11 antibodies specificallyrecognize pathological IAPP amyloid in the pancreas of patientsdiagnosed with diabetes mellitus type 2 (T2D). NI-203.9A2, NI-203.19H8and NI-203.26C11 antibodies show a staining in T2D pancreatic isletsloaded with IAPP fibrils (amyloid) (A, B) but not in T2D pancreaticislets lacking IAPP fibrils (C, D). (A) Thioflavin S (ThioS, left panel)and Congo red (CR, right panel) staining of amyloid in pancreatic isletsof a T2D patient. (B) Detection of IAPP fibrils on amyloid positive T2Dpancreatic islets with NI-203.9A2, NI-203.19H8 and NI-203.26C11antibodies (brown-CR) at 50 nM (large panel and bottom left inset) and 5nM (bottom right inset). (C) Absence of amyloid in pancreatic islets ofa T2D patient, as shown by negative thioflavin S (ThioS, left panel) andCongo red (CR, right panel) staining. (D) Absence of staining on amyloidnegative T2D pancreatic islets with NI-203.9A2, NI-203.19H8 andNI-203.26C11 antibodies at 50 nM. Secondary donkey anti-human antibodyonly (IIary Ab) was used as a control. Bottom insets: high magnificationimages of individual human pancreatic islets. Human pancreatic isletswere stained with anti-insulin antibody (blue in original (i.o.), strongstaining here) and counterstaining was performed to visualize cellnuclei (faint blue i.o., faint staining here).

FIG. 8: NI-203.9A2, NI-203.19H8 and NI-203.26C11 antibodies do notrecognize physiological IAPP on human control pancreas. NI-203.9A2,NI-203.19H8 and NI-203.26C11 antibodies (50 nM) show weak staining onhuman control islets when compared to the IAPP control antibody (1:100;control Ab). Secondary donkey anti-human antibody only (IIary Ab) wasused as a control. Human pancreatic islets were stained withanti-insulin antibody (blue in original (i.o.), strong staining here)and counterstaining was performed to visualize cell nuclei (faint bluei.o., faint staining here).

FIG. 9: NI-203.9A2, NI-203.19H8 and NI-203.26C11 antibodies recognizepathological IAPP fibrils on a diabetic cat pancreas. Detection of IAPPfibrils on pancreatic islets of a T2D cat with NI-203.9A2, NI-203.19H8and NI-203.26C11 antibodies (50 nM, brown-CR). IAPP fibrils (amyloid)were stained with Congo red (CR). Secondary donkey anti-human antibodyonly (IIary Ab) was used as a control. Bottom left insets: highmagnification images of individual cat pancreatic islets. Cat pancreaticislets were stained with anti-insulin antibody (blue in original (i.o.),strong staining here) and counterstaining was performed to visualizecell nuclei (faint blue i.o., faint staining here).

FIG. 10: NI-203.9A2, NI-203.19H8 and NI-203.26C11 antibodies do notrecognize pathological Aβ deposits in a human brain with Alzheimer'sdisease. Absence of staining with NI-203.9A2, NI-203.19H8 andNI-203.26C11 antibodies (50 nM), in opposition to the AP-specificantibody 6E10 (1:2000; control Ab). Secondary donkey anti-human anibodyonly (IIary Ab) was used as a control. Counterstaining was performed tovisualize cell nuclei (faint blue i.o., faint staining here).

FIG. 11: Recombinant human and mouse chimeric antibody NI-203.9A2,NI-203.19H8 and NI-203.26C11 bind with equal affinity to human IAPP. (A)EC₅₀ determination of the recombinant human and mouse chimeric anti-IAPPantibodies for IAPP (□, 10 μg/ml) and BSA (⋄, 10 μg/ml). Plates wereincubated with the indicated concentrations of antibodies. Measurementswere made in duplicate. Data are expressed as mean OD values at 450 nm.(B, C) EC₅₀ values of human and mouse chimeric antibodies.

FIG. 12: Recombinant mouse chimeric antibody NI-203.9A2, NI-203.19H8 andNI-203.26C11 recognize pathological IAPP fibrils in the pancreas ofpatients diagnosed with diabetes mellitus type 2 (T2D). Detection ofIAPP fibrils on pancreatic islets of two human T2D patients (1 and 2)with chimeric NI-203.9A2, NI-203.19H8 and NI-203.26C11 antibodies at 50nM (brown i.o., strong dark to black staining here). Human pancreaticislets were stained with anti-insulin antibody (blue i.o., strongstaining here) and counterstaining was performed to visualize cellnuclei (faint blue i.o., faint staining here).

DETAILED DESCRIPTION OF THE INVENTION

In type-2 diabetes (T2D) genetic determinants and environmental factorslead to the development of insulin resistance followed by a compensatoryincrease in beta-cell mass and insulin and amylin (hIAPP) secretion tomaintain normal blood glucose levels. The resulting high concentrationsof amylin favor the formation of toxic human islet amyloid polypeptide(hIAPP) oligomers and deposition of hIAPP fibrils which is found in morethan 90% of type-2 diabetes patients. The deposition of hIAPP correlateswith the reduction in insulin producing beta-cells and has also beenproposed to play a role for the loss of β-cells in pancreatic isletstransplanted into individuals with type-1 diabetes. Severalhuman-derived antibodies from pools of healthy or obese donors with highrisk for type-2 diabetes but absence of disease have been identified andcharacterized in vitro, cloned and produced recombinantly byNeurimmune's RTM™ technology as described in detail in internationalapplication WO2008/081008 and are provided herein. Lead candidates arevalidated in transgenic mice expressing hIAPP and exposed to high fatdiet. Therapeutic efficacy is assessed by determining the beta-cell massand hIAPP amyloid load in the pancreas as well as plasma levels ofhIAPP, and functional tests of glucose metabolism and insulin secretion.

Type-2 diabetes is the most common form of diabetes, accounting forabout 90% of all cases. The disease affects more than 200 million peopleworldwide resulting in more than a million deaths from diabetesannually. More than 300.000 patients are affected in Switzerland. Theprevalence of diabetes is increasing dramatically in both developed anddeveloping countries due to population growth, aging, urbanization, andincreasing prevalence of obesity and physical inactivity. The globaltype-2 diabetes market at USD 25 billion is forecast to reach USD 35billion by 2016 with a compound annual growth rate of 6.4% between 2009and 2016. Current treatments include dietary management andpharmacological intervention acting on different pathways to decreaseblood glucose levels by either improving insulin sensitivity orstimulating the pancreas to release insulin. None of the availabletreatments can however counteract the aggregation of hIAPP and the lossof pancreatic beta-cells. New treatment strategies for type-2 diabetesinvolve analogues of glucagon-peptide 1 (GLP-1) and inhibitors ofdipeptidyl-peptidase 4 (DPP 4), the enzyme which inactivates endogenousGLP-1. These strategies are based on the potent insulinotropic effect ofGLP-1 and its effect to enhance beta-cell proliferation. Importantly,increased insulin release is also coupled to increased amylin release.Experimentally, stimulated insulin secretion has been shown to promotethe development of islet amyloidosis in animal models and similareffects can be expected in humans. Besides the particular use of IAPPbinding molecules of the present invention a further proposedtherapeutic approach might therefore be an attractive combinationtherapy of these molecules and the above indicated novel treatments.

I. Definitions

Unless otherwise stated, a term as used herein is given the definitionas provided in the Oxford Dictionary of Biochemistry and MolecularBiology, Oxford University Press, 1997, revised 2000 and reprinted 2003,ISBN 0 19 850673 2.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an antibody,” is understood to representone or more antibodies. As such, the terms “a” (or “an”), “one or more,”and “at least one” can be used interchangeably herein.

If not specifically indicated otherwise, the term “IAPP”, is usedinterchangeable to specifically refer to the native monomeric,oligomeric, non-fibrillar and fibrillar form of islet amyloidpolypeptide (IAPP). The term “IAPP” is also used to generally identifyother conformers of IAPP, for example, oligomers and/or aggregates ofIAPP such as IAPP-fibrils. The term “IAPP” is also used to refercollectively to all types and forms of IAPP. The term proIAPP is usedinterchangeable to specifically refer to the native monomeric,oligomeric, fibrillar and/or aggregated form of the precursor peptide ofthe islet amyloid polypeptide (proIAPP). Added letters in front of theterms IAPP or proIAPP are used to indicate the organism the particularortholog is originating from, e.g. hIAPP for human IAPP or mIAPP formurine origin.

The amino acid sequence of 37 aa for human IAPP is:KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY (SEQ ID NO: 1) with a disulfidebridge between cysteine residues 2 and 7 and an amidated C-terminus.

IAPP is processed from preprohormone preproIAPP, a 89 amino acidprecursor produced in pancreatic β-cells. The protein sequence for humanpreproIAPP is: MGILKLQVFLIVLSVALNHLKATPIESHQVEKRKCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTYGKRNAVEVLKREPLNYLPL (SEQ ID NO: 2), which sequence may befound as well in pertinent databases, e.g., in the UniProt database:UniProtID: P10997 (IAPP_HUMAN).

PreproIAPP is rapidly cleaved after translation into proislet amyloidpolypeptide. The protein sequence for human proIAPP is:TPIESHQVEKRKCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTYGKRNAVEV LKREPLNYLPL(SEQ ID NO: 3), which undergoes additional proteolysis andpost-translational modifications to generate hIAPP.

The “wild type” or recombinant human IAPP, proIAPP and preproIAPP aminoacid sequences are represented by the above mentioned sequencesaccording to SEQ ID NOs: 1-3.

The human anti-IAPP and anti-proIAPP antibodies disclosed hereinspecifically bind IAPP and/or proIAPP and epitopes thereof and tovarious conformations of IAPP and/or proIAPP and epitopes thereof. Forexample, disclosed herein are antibodies that specifically bindpathologically aggregated IAPP and/or proIAPP forms, such asnon-fibrillar oligomers and/or fibrillar oligomers/fibrils and/oraggregates consisting of mixed forms thereof. The term (pathologically)aggregated/aggregates of IAPP and/or proIAPP is used interchangeable tospecifically refer to the aforementioned forms. The term (pathological)“aggregated forms” or “aggregates” as used herein describes the productsof an accumulation or cluster formation due to an IAPP and/or proIAPPerroneous/pathological interaction with one another. These aggregates,accumulations or cluster forms may be, substantially consist or consistof both IAPP and/or proIAPP and of non-fibrillar oligomers and/orfibrillar oligomers and fibrils thereof. As used herein, reference to anantibody that “specifically binds”, “selectively binds”, or“preferentially binds” IAPP and/or proIAPP refers to an antibody thatdoes not bind other unrelated proteins. In one example, an IAPP and/orproIAPP antibody disclosed herein can bind IAPP and/or proIAPP or anepitope thereof and show no binding above about 2 times background forother proteins. An antibody that “specifically binds” or “selectivelybinds” an IAPP and/or proIAPP conformer refers to an antibody that doesnot bind all conformations of IAPP and/or proIAPP, i.e., does not bindat least one other IAPP and/or proIAPP conformer. For example, disclosedherein are antibodies that can preferentially bind to aggregated formsof IAPP and/or proIAPP both in vitro and in tissues obtained frompatients with overt T2D or with a risk to develop T2D. Since the humanIAPP and/or proIAPP antibodies of the present invention have beenisolated from a pool of healthy human subjects or from pools of obesepatients and other patients groups with enhanced risk to develop T2D,which at the time of antibody isolation showed no signs of T2D,exhibiting an IAPP and/or proIAPP specific immune response, the IAPPand/or proIAPP antibodies of the present invention may also be called“human auto-antibodies” in order to emphasize that those antibodies wereindeed expressed by the subjects and have not been isolated from, forexample a human immunoglobulin expressing phage library, which hithertorepresented one common method for trying to provide human-likeantibodies.

The term “peptide” is understood to include the terms “polypeptide” and“protein” (which, at times, may be used interchangeably herein) withinits meaning. Similarly, fragments of proteins and polypeptides are alsocontemplated and may be referred to herein as “peptides”. Nevertheless,the term “peptide” preferably denotes an amino acid polymer including atleast 5 contiguous amino acids, preferably at least 10 contiguous aminoacids, more preferably at least 15 contiguous amino acids, still morepreferably at least 20 contiguous amino acids, and particularlypreferred at least 25 contiguous amino acids. In addition, the peptidein accordance with present invention typically has no more than 100contiguous amino acids, preferably less than 80 contiguous amino acidsand more preferably less than 50 contiguous amino acids.

Polypeptides:

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, “peptides,” “dipeptides,”“tripeptides, “oligopeptides,” “protein,” “amino acid chain,” or anyother term used to refer to a chain or chains of two or more aminoacids, are included within the definition of “polypeptide,” and the term“polypeptide” may be used instead of, or interchangeably with any ofthese terms.

The term “polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation andderivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

A polypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid residue, e.g., a serine residue or an asparagineresidue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

“Recombinant peptides, polypeptides or proteins” refer to peptides,polypeptides or proteins produced by recombinant DNA techniques, i.e.produced from cells, microbial or mammalian, transformed by an exogenousrecombinant DNA expression construct encoding the fusion proteinincluding the desired peptide. Proteins or peptides expressed in mostbacterial cultures will typically be free of glycan. Proteins orpolypeptides expressed in yeast may have a glycosylation patterndifferent from that expressed in mammalian cells.

Included as polypeptides of the present invention are fragments,derivatives, analogs or variants of the foregoing polypeptides and anycombinations thereof as well. The terms “fragment,” “variant,”“derivative” and “analog” include peptides and polypeptides having anamino acid sequence sufficiently similar to the amino acid sequence ofthe natural peptide. The term “sufficiently similar” means a first aminoacid sequence that contains a sufficient or minimum number of identicalor equivalent amino acid residues relative to a second amino acidsequence such that the first and second amino acid sequences have acommon structural domain and/or common functional activity. For example,amino acid sequences that comprise a common structural domain that is atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or at least about 100%, identical are definedherein as sufficiently similar. Preferably, variants will besufficiently similar to the amino acid sequence of the preferredpeptides of the present invention, in particular to IAPP and/or proIAPPor fragments, variants, derivatives or analogs of either of them. Suchvariants generally retain the functional activity of the peptides of thepresent invention. Variants include peptides that differ in amino acidsequence from the native and wt peptide, respectively, by way of one ormore amino acid deletion(s), addition(s), and/or substitution(s). Thesemay be naturally occurring variants as well as artificially designedones.

Furthermore, the terms “fragment,” “variant,” “derivative” and “analog”when referring to antibodies or antibody polypeptides of the presentinvention include any polypeptides which retain at least some of theantigen-binding properties of the corresponding native binding molecule,antibody, or polypeptide. Fragments of polypeptides of the presentinvention include proteolytic fragments, as well as deletion fragments,in addition to specific antibody fragments discussed elsewhere herein.Variants of antibodies and antibody polypeptides of the presentinvention include fragments as described above, and also polypeptideswith altered amino acid sequences due to amino acid substitutions,deletions, or insertions. Variants may occur naturally or benon-naturally occurring. Non-naturally occurring variants may beproduced using art-known mutagenesis techniques. Variant polypeptidesmay comprise conservative or non-conservative amino acid substitutions,deletions or additions. Derivatives of IAPP and/or proIAPP specificbinding molecules, e.g., antibodies and antibody polypeptides of thepresent invention, are polypeptides which have been altered so as toexhibit additional features not found on the native polypeptide.Examples include fusion proteins. Variant polypeptides may also bereferred to herein as “polypeptide analogs”. As used herein a“derivative” of a binding molecule or fragment thereof, an antibody, oran antibody polypeptide refers to a subject polypeptide having one ormore residues chemically derivatized by reaction of a functional sidegroup. Also included as “derivatives” are those peptides which containone or more naturally occurring amino acid derivatives of the twentystandard amino acids. For example, 4-hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.

Determination of Similarity and/or Identity of Molecules:

“Similarity” between two peptides is determined by comparing the aminoacid sequence of one peptide to the sequence of a second peptide. Anamino acid of one peptide is similar to the corresponding amino acid ofa second peptide if it is identical or a conservative amino acidsubstitution. Conservative substitutions include those described inDayhoff, M. O., ed., The Atlas of Protein Sequence and Structure 5,National Biomedical Research Foundation, Washington, D.C. (1978), and inArgos, EMBO J. 8 (1989), 779-785. For example, amino acids belonging toone of the following groups represent conservative changes orsubstitutions: -Ala, Pro, Gly, Gln, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr;-Val, Ile, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and-Asp, Glu.

“Similarity” between two polynucleotides is determined by comparing thenucleic acid sequence of one polynucleotide to the sequence of apolynucleotide. A nucleic acid of one polynucleotide is similar to thecorresponding nucleic acid of a second polynucleotide if it is identicalor, if the nucleic acid is part of a coding sequence, the respectivetriplet comprising the nucleic acid encodes for the same amino acid orfor a conservative amino acid substitution.

The determination of percent identity or similarity between twosequences is preferably accomplished using the mathematical algorithm ofKarlin and Altschul (1993) Proc. Natl. Acad. Sci USA 90: 5873-5877. Suchan algorithm is incorporated into the BLASTn and BLASTp programs ofAltschul et al. (1990) J. Mol. Biol. 215: 403-410 available at NCBI(http://www.ncbi.nlmnih.gov/blast/Blast.cge).

The determination of percent identity or similarity is performed withthe standard parameters of the BLASTn and BLASTp programs, asrecommended on the NCBI webpage and in the “BLAST Program SelectionGuide” in respect of sequences of a specific length and composition.

BLAST polynucleotide searches are performed with the BLASTn program.

For the general parameters, the “Max Target Sequences” box may be set to100, the “Short queries” box may be ticked, the “Expect threshold” boxmay be set to 1000 and the “Word Size” box may be set to 7 asrecommended for short sequences (less than 20 bases) on the NCBIwebpage. For longer sequences the “Expect threshold” box may be set to10 and the “Word Size” box may be set to 11. For the scoring parametersthe “Match/mismatch Scores” may be set to 1,-2 and the “Gap Costs” boxmay be set to linear. For the Filters and Masking parameters, the “Lowcomplexity regions” box may not be ticked, the “Species-specificrepeats” box may not be ticked, the “Mask for lookup table only” box maybe ticked, the “DUST Filter Settings” may be ticked and the “Mask lowercase letters” box may not be ticked. In general the “Search for shortnearly exact matches” may be used in this respect, which provides mostof the above indicated settings. Further information in this respect maybe found in the “BLAST Program Selection Guide” published on the NCBIwebpage.

BLAST protein searches are performed with the BLASTp program. For thegeneral parameters, the “Max Target Sequences” box may be set to 100,the “Short queries” box may be ticked, the “Expect threshold” box may beset to 10 and the “Word Size” box may be set to “3”. For the scoringparameters the “Matrix” box may be set to “BLOSUM62”, the “Gap Costs”Box may be set to “Existence: 11 Extension: 1”, the “Compositionaladjustments” box may be set to “Conditional compositional score matrixadjustment”. For the Filters and Masking parameters the “Low complexityregions” box may not be ticked, the “Mask for lookup table only” box maynot be ticked and the “Mask lower case letters” box may not be ticked.

Modifications of both programs, e.g., in respect of the length of thesearched sequences, are performed according to the recommendations inthe “BLAST Program Selection Guide” published in a HTML and a PDFversion on the NCBI webpage.

Polynucleotides:

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide may comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The term “nucleic acid” refers to any oneor more nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide. By “isolated” nucleic acid or polynucleotide is intendeda nucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encodingan antibody contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, polynucleotide or a nucleic acid may be or may include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding abinding molecule, an antibody, or fragment, variant, or derivativethereof. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid whichencodes a polypeptide normally may include a promoter and/or othertranscription or translation control elements operable associated withone or more coding regions. An operable association is when a codingregion for a gene product, e.g., a polypeptide, is associated with oneor more regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operable associated” or “operablelinked” if induction of promoter function results in the transcriptionof mRNA encoding the desired gene product and if the nature of thelinkage between the two DNA fragments does not interfere with theability of the expression regulatory sequences to direct the expressionof the gene product or interfere with the ability of the DNA template tobe transcribed. Thus, a promoter region would be operable associatedwith a nucleic acid encoding a polypeptide if the promoter was capableof effecting transcription of that nucleic acid. The promoter may be acell-specific promoter that directs substantial transcription of the DNAonly in predetermined cells. Other transcription control elements,besides a promoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operable associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal peptideor secretory leader sequence which is cleaved from the mature proteinonce export of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that polypeptides secreted by vertebrate cells generally have asignal peptide fused to the N-terminus of the polypeptide, which iscleaved from the complete or “full-length” polypeptide to produce asecreted or “mature” form of the polypeptide. In certain embodiments,the native signal peptide, e.g., an immunoglobulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operable associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse β-glucuronidase.

A “binding molecule” as used in the context of the present inventionrelates primarily to antibodies, and fragments thereof, but may alsorefer to other non-antibody molecules that bind to IAPP and/or proIAPPincluding but not limited to hormones, receptors, ligands, majorhistocompatibility complex (MHC) molecules, chaperones such as heatshock proteins (HSPs) as well as cell-cell adhesion molecules such asmembers of the cadherin, intergrin, C-type lectin and immunoglobulin(Ig) superfamilies. Thus, for the sake of clarity only and withoutrestricting the scope of the present invention most of the followingembodiments are discussed with respect to antibodies and antibody-likemolecules which represent the preferred binding molecules for thedevelopment of therapeutic and diagnostic agents.

Antibodies:

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin is an IAPP and/or proIAPP-bindingmolecule which comprises at least the variable domain of a heavy chain,and normally comprises at least the variable domains of a heavy chainand a light chain. Basic immunoglobulin structures in vertebrate systemsare relatively well understood; see, e.g., Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is thenature of this chain that determines the “class” of the antibody as IgG,IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses(isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc. are wellcharacterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernible to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of the instant invention. Allimmunoglobulin classes are clearly within the scope of the presentinvention, the following discussion will generally be directed to theIgG class of immunoglobulin molecules. With regard to IgG, a standardimmunoglobulin molecule comprises two identical light chain polypeptidesof molecular weight approximately 23,000 Daltons, and two identicalheavy chain polypeptides of molecular weight 53,000-70,000. The fourchains are typically joined by disulfide bonds in a “Y” configurationwherein the light chains bracket the heavy chains starting at the mouthof the “Y” and continuing through the variable region.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (V_(L)) and heavy (V_(H)) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3)confer important biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen-binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the V_(L) domain and V_(H) domain, or subset of the complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three dimensional antigen-binding site. Thisquaternary antibody structure forms the antigen-binding site present atthe end of each arm of the Y. More specifically, the antigen-bindingsite is defined by three CDRs on each of the V_(H) and V_(L) chains. Anyantibody or immunoglobulin fragment which contains sufficient structureto specifically bind to IAPP and/or proIAPP is denoted hereininterchangeably as a “binding fragment” or an “immunospecific fragment.”

In naturally occurring antibodies, an antibody comprises sixhypervariable regions, sometimes called “complementarity determiningregions” or “CDRs” present in each antigen-binding domain, which areshort, non-contiguous sequences of amino acids that are specificallypositioned to form the antigen-binding domain as the antibody assumesits three dimensional configuration in an aqueous environment. The“CDRs” are flanked by four relatively conserved “framework” regions or“FRs” which show less inter-molecular variability. The framework regionslargely adopt a β-sheet conformation and the CDRs form loops whichconnect, and in some cases form part of, the β-sheet structure. Thus,framework regions act to form a scaffold that provides for positioningthe CDRs in correct orientation by inter-chain, non-covalentinteractions. The antigen-binding domain formed by the positioned CDRsdefines a surface complementary to the epitope on the immunoreactiveantigen. This complementary surface promotes the non-covalent binding ofthe antibody to its cognate epitope. The amino acids comprising the CDRsand the framework regions, respectively, can be readily identified forany given heavy or light chain variable region by one of ordinary skillin the art, since they have been precisely defined; see, “Sequences ofProteins of Immunological Interest,” Kabat, E., et al., U.S. Departmentof Health and Human Services, (1983); and Chothia and Lesk, J. Mol.Biol., 196 (1987), 901-917, which are incorporated herein by referencein their entireties.

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaand Lesk, J. Mol. Biol., 196 (1987), 901-917, which are incorporatedherein by reference, where the definitions include overlapping orsubsets of amino acid residues when compared against each other.Nevertheless, application of either definition to refer to a CDR of anantibody or variants thereof is intended to be within the scope of theterm as defined and used herein. The appropriate amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth below in Table I as a comparison. The exactresidue numbers which encompass a particular CDR will vary depending onthe sequence and size of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular hypervariable region orCDR of the human IgG subtype of antibody given the variable region aminoacid sequence of the antibody.

TABLE I CDR Definitions¹ Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-6552-58 VH CDR3 95-102 95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VLCDR3 89-97 91-96 ¹Numbering of all CDR definitions in Table I isaccording to the numbering conventions set forth by Kabat et al. (seebelow).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody or antigen-binding fragment,variant, or derivative thereof of the present invention are according tothe Kabat numbering system, which however is theoretical and may notequally apply to every antibody of the present invention. For example,depending on the position of the first CDR the following CDRs might beshifted in either direction.

Antibodies or antigen-binding fragments, immunospecific fragments,variants, or derivatives thereof of the invention include, but are notlimited to, polyclonal, monoclonal, multispecific, human, humanized,primatized, murinized or chimeric antibodies, single chain antibodies,epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)₂, Fd, Fvs,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv), fragments comprising either a V_(L) or V_(H) domain, fragmentsproduced by a Fab expression library, and anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies disclosedherein). ScFv molecules are known in the art and are described, e.g., inU.S. Pat. No. 5,892,019 Immunoglobulin or antibody molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule.

In one embodiment, the antibody of the present invention is not IgM or aderivative thereof with a pentavalent structure. Particular, in specificapplications of the present invention, especially therapeutic use, IgMsare less useful than IgG and other bivalent antibodies or correspondingbinding molecules since IgMs due to their pentavalent structure and lackof affinity maturation often show unspecific cross-reactivities and verylow affinity.

In a particularly preferred embodiment, the antibody of the presentinvention is not a polyclonal antibody, i.e. it substantially consistsof one particular antibody species rather than being a mixture obtainedfrom a plasma immunoglobulin sample.

Antibody fragments, including single-chain antibodies, may comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, CH1, CH2, and CH3 domains. Alsoincluded in the invention are IAPP and/or proIAPP binding fragmentswhich comprise any combination of variable region(s) with a hingeregion, CH1, CH2, and CH3 domains. Antibodies or immunospecificfragments thereof of the present invention may be from any animal originincluding birds and mammals. Preferably, the antibodies are human,murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, orchicken antibodies. In another embodiment, the variable region may becondricthoid in origin (e.g., from sharks).

In one aspect, the antibody of the present invention is a humanmonoclonal antibody isolated from a human. Optionally, the frameworkregion of the human antibody is aligned and adopted in accordance withthe pertinent human germ line variable region sequences in the database;see, e.g., Vbase (http://vbase.mrc-cpe.cam.ac.uk/) hosted by the MRCCentre for Protein Engineering (Cambridge, UK). For example, amino acidsconsidered to potentially deviate from the true germ line sequence couldbe due to the PCR primer sequences incorporated during the cloningprocess. Compared to artificially generated human-like antibodies suchas single chain antibody fragments (scFvs) from a phage displayedantibody library or xenogeneic mice the human monoclonal antibody of thepresent invention is characterized by (i) being obtained using the humanimmune response rather than that of animal surrogates, i.e. the antibodyhas been generated in response to natural IAPP or proIAPP in itsrelevant conformation in the human body, (ii) having protected theindividual or is at least significant for the presence of IAPP and/orproIAPP, and (iii) since the antibody is of human origin the risks ofcross-reactivity against self-antigens is minimized Thus, in accordancewith the present invention the terms “human monoclonal antibody”, “humanmonoclonal autoantibody”, “human antibody” and the like are used todenote an IAPP and/or proIAPP binding molecule which is of human origin,i.e. which has been isolated from a human cell such as a B cell orhybridoma thereof or the cDNA of which has been directly cloned frommRNA of a human cell, for example a human memory B cell. A humanantibody is still “human” even if amino acid substitutions are made inthe antibody, e.g., to improve binding characteristics.

Antibodies derived from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described infra and, for example in, U.S.Pat. No. 5,939,598 by Kucherlapati et al., are denoted human-likeantibodies in order distinguish them from truly human antibodies of thepresent invention.

For example, the paring of heavy and light chains of human-likeantibodies such as synthetic and semi-synthetic antibodies typicallyisolated from phage display do not necessarily reflect the originalparing as it occurred in the original human B cell. Accordingly Fab andscFv fragments obtained from recombinant expression libraries ascommonly used in the prior art can be considered as being artificialwith all possible associated effects on immunogenicity and stability.

In contrast, the present invention provides isolated affinity-maturedantibodies from selected human subjects, which are characterized bytheir therapeutic utility and their tolerance in man.

As used herein, the term “rodentized antibody” or “rodentizedimmunoglobulin” refers to an antibody comprising one or more CDRs from ahuman antibody of the present invention; and a human framework regionthat contains amino acid substitutions and/or deletions and/orinsertions that are based on a rodent antibody sequence. When referredto rodents, preferably sequences originating in mice and rats are used,wherein the antibodies comprising such sequences are referred to as“murinized” or “ratinized” respectively. The human immunoglobulinproviding the CDRs is called the “parent” or “acceptor” and the rodentantibody providing the framework changes is called the “donor”. Constantregions need not be present, but if they are, they are usuallysubstantially identical to the rodent antibody constant regions, i.e. atleast about 85-90%, preferably about 95% or more identical. Hence, insome embodiments, a full-length murinized human heavy or light chainimmunoglobulin contains a mouse constant region, human CDRs, and asubstantially human framework that has a number of “murinizing” aminoacid substitutions. Typically, a “murinized antibody” is an antibodycomprising a murinized variable light chain and/or a murinized variableheavy chain. For example, a murinized antibody would not encompass atypical chimeric antibody, e.g., because the entire variable region of achimeric antibody is non-mouse. A modified antibody that has been“murinized” by the process of “murinization” binds to the same antigenas the parent antibody that provides the CDRs and is usually lessimmunogenic in mice, as compared to the parent antibody. The aboveexplanations in respect of “murinized” antibodies apply analogously foroder “rodentized” antibodies, such as “ratinized antibodies”, whereinrat sequences are used instead of the murine.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a CH1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide for use in the invention may comprise apolypeptide chain comprising a CH1 domain; a polypeptide chaincomprising a CH1 domain, at least a portion of a hinge domain, and a CH2domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; apolypeptide chain comprising a CH1 domain, at least a portion of a hingedomain, and a CH3 domain, or a polypeptide chain comprising a CH1domain, at least a portion of a hinge domain, a CH2 domain, and a CH3domain. In another embodiment, a polypeptide of the invention comprisesa polypeptide chain comprising a CH3 domain. Further, a bindingpolypeptide for use in the invention may lack at least a portion of aCH2 domain (e.g., all or part of a CH2 domain). As set forth above, itwill be understood by one of ordinary skill in the art that thesedomains (e.g., the heavy chain portions) may be modified such that theyvary in amino acid sequence from the naturally occurring immunoglobulinmolecule.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein, the heavy chain portions of onepolypeptide chain of a multimer are identical to those on a secondpolypeptide chain of the multimer. Alternatively, heavy chainportion-containing monomers of the invention are not identical. Forexample, each monomer may comprise a different target binding site,forming, for example, a bispecific antibody or diabody.

In another embodiment, the antibodies, or antigen-binding fragments,variants, or derivatives thereof disclosed herein are composed of asingle polypeptide chain such as scFvs and are to be expressedintracellularly (intrabodies) for potential in vivo therapeutic anddiagnostic applications.

The heavy chain portions of a binding polypeptide for use in thediagnostic and treatment methods disclosed herein may be derived fromdifferent immunoglobulin molecules. For example, a heavy chain portionof a polypeptide may comprise a CH1 domain derived from an IgG1 moleculeand a hinge region derived from an IgG3 molecule. In another example, aheavy chain portion can comprise a hinge region derived, in part, froman IgG1 molecule and, in part, from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. Preferably, thelight chain portion comprises at least one of a V_(L) or CL domain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes preferably contain at least seven, more preferably at leastnine and most preferably between at least about 15 to about 30 aminoacids. Since a CDR can recognize an antigenic peptide or polypeptide inits tertiary form, the amino acids comprising an epitope need not becontiguous, and in some cases, may not even be on the same peptidechain. In the present invention, a peptide or polypeptide epitoperecognized by antibodies of the present invention contains a sequence ofat least 4, at least 5, at least 6, at least 7, more preferably at least8, at least 9, at least 10, at least 15, at least 20, at least 25, orbetween about 15 to about 30 contiguous or non-contiguous amino acids ofIAPP or proIAPP.

By “specifically binding”, or “specifically recognizing”, usedinterchangeably herein, it is generally meant that a binding molecule,e.g., an antibody binds to an epitope via its antigen-binding domain,and that the binding entails some complementarity between theantigen-binding domain and the epitope. According to this definition, anantibody is said to “specifically bind” to an epitope when it binds tothat epitope, via its antigen-binding domain more readily than it wouldbind to a random, unrelated epitope. The term “specificity” is usedherein to qualify the relative affinity by which a certain antibodybinds to a certain epitope. For example, antibody “A” may be deemed tohave a higher specificity for a given epitope than antibody “B,” orantibody “A” may be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D”.

Where present, the term “immunological binding characteristics,” orother binding characteristics of an antibody with an antigen, in all ofits grammatical forms, refers to the specificity, affinity,cross-reactivity, and other binding characteristics of an antibody.

By “preferentially binding”, it is meant that the binding molecule,e.g., antibody specifically binds to an epitope more readily than itwould bind to a related, similar, homologous, or analogous epitope.Thus, an antibody which “preferentially binds” to a given epitope wouldmore likely bind to that epitope than to a related epitope, even thoughsuch an antibody may cross-react with the related epitope.

By way of non-limiting example, a binding molecule, e.g., an antibodymay be considered to bind a first epitope preferentially if it bindssaid first epitope with a dissociation constant (K_(D)) that is lessthan the antibody's K_(D) for the second epitope. In anothernon-limiting example, an antibody may be considered to bind a firstantigen preferentially if it binds the first epitope with an affinitythat is at least one order of magnitude less than the antibody's K_(D)for the second epitope. In another non-limiting example, an antibody maybe considered to bind a first epitope preferentially if it binds thefirst epitope with an affinity that is at least two orders of magnitudeless than the antibody's K_(D) for the second epitope.

In another non-limiting example, a binding molecule, e.g., an antibodymay be considered to bind a first epitope preferentially if it binds thefirst epitope with an off rate (k(off)) that is less than the antibody'sk(off) for the second epitope. In another non-limiting example, anantibody may be considered to bind a first epitope preferentially if itbinds the first epitope with an affinity that is at least one order ofmagnitude less than the antibody's k(off) for the second epitope. Inanother non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds the first epitope with anaffinity that is at least two orders of magnitude less than theantibody's k(off) for the second epitope.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein may be said to bind IAPP and/orproIAPP or a fragment, variant or specific conformation thereof with anoff rate (k(off)) of less than or equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹,5×10⁻³ sec⁻¹ or 10⁻³ sec⁻¹. More preferably, an antibody of theinvention may be said to bind IAPP and/or proIAPP or a fragment, variantor specific conformation thereof with an off rate (k(off)) less than orequal to 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹ 5×10⁻⁶sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻⁷ or 10⁻⁷ sec⁻¹.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein may be said to bind IAPP and/orproIAPP or a fragment, variant or specific conformation thereof with anon rate (k(on)) of greater than or equal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹sec⁻¹, 10⁴ M⁻¹ sec⁻¹ or 5×10⁴ M⁻¹ sec⁻¹. More preferably, an antibody ofthe invention may be said to bind IAPP and/or proIAPP or a fragment,variant or specific conformation thereof with an on rate (k(on)) greaterthan or equal to 10⁻⁵ M⁻¹ sec⁻¹, 5×10⁻⁵ M⁻¹ sec⁻¹, 10⁻⁶ M⁻¹ sec⁻¹, or5×10⁻⁶ M⁻¹ sec⁻¹ or 10⁻⁷ M⁻¹ sec⁻¹.

A binding molecule, e.g., an antibody is said to competitively inhibitbinding of a reference antibody to a given epitope if it preferentiallybinds to that epitope to the extent that it blocks, to some degree,binding of the reference antibody to the epitope. Competitive inhibitionmay be determined by any method known in the art, for example,competition ELISA assays. An antibody may be said to competitivelyinhibit binding of the reference antibody to a given epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of a bindingmolecule, e.g., an immunoglobulin molecule; see, e.g., Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,2nd ed. (1988) at pages 27-28. As used herein, the term “avidity” refersto the overall stability of the complex between a population ofimmunoglobulins and an antigen, that is, the functional combiningstrength of an immunoglobulin mixture with the antigen; see, e.g.,Harlow at pages 29-34. Avidity is related to both the affinity ofindividual immunoglobulin molecules in the population with specificepitopes, and also the valences of the immunoglobulins and the antigen.For example, the interaction between a bivalent monoclonal antibody andan antigen with a highly repeating epitope structure, such as a polymer,would be one of high avidity. The affinity or avidity of an antibody foran antigen can be determined experimentally using any suitable method;see, for example, Berzofsky et al., “Antibody-Antigen Interactions” InFundamental Immunology, Paul, W. E., Ed., Raven Press New York, N Y(1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, N Y(1992), and methods described herein. General techniques for measuringthe affinity of an antibody for an antigen include ELISA, RIA, andsurface plasmon resonance. The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions, e.g., salt concentration, pH. Thus, measurements of affinityand other antigen-binding parameters, e.g., K_(D), IC₅₀, are preferablymade with standardized solutions of antibody and antigen, and astandardized buffer.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their cross-reactivity. As used herein, theterm “cross-reactivity” refers to the ability of an antibody, specificfor one antigen, to react with a second antigen; a measure ofrelatedness between two different antigenic substances. Thus, anantibody is cross reactive if it binds to an epitope other than the onethat induced its formation. The cross reactive epitope generallycontains many of the same complementary structural features as theinducing epitope, and in some cases, may actually fit better than theoriginal.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes, e.g., epitopes withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be said to have littleor no cross-reactivity if it does not bind epitopes with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be deemed “highlyspecific” for a certain epitope, if it does not bind any other analog,ortholog, or homolog of that epitope.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their binding affinity to IAPP and/or proIAPP.Preferred binding affinities include those with a dissociation constantor Kd less than 5×10⁻² M, 10⁻³ M, 5×10⁻³M, 10⁻³M, 5×10⁻⁴M, 10⁻⁴M,5×10⁻⁵M, 10⁻⁵M, 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M,10⁻⁹M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹²M, 10⁻¹²M,5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “V_(H) domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CH1 domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CH1 domain isadjacent to the V_(H) domain and is amino terminal to the hinge regionof an immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat E A et al. op. cit). The CH2 domain is unique in that it isnot closely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two CH2 domains of anintact native IgG molecule. It is also well documented that the CH3domain extends from the CH2 domain to the C-terminal of the IgG moleculeand comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen-binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains; see Roux et al., J.Immunol. 161 (1998), 4083-4090.

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CL regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

As used herein, the terms “linked”, “fused” or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. An “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two or more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature). Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence. Forexample, polynucleotides encoding the CDRs of an immunoglobulin variableregion may be fused, in-frame, but be separated by a polynucleotideencoding at least one immunoglobulin framework region or additional CDRregions, as long as the “fused” CDRs are co-translated as part of acontinuous polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, an RNA or polypeptide. The processincludes any manifestation of the functional presence of the gene withinthe cell including, without limitation, gene knockdown as well as bothtransient expression and stable expression. It includes withoutlimitation transcription of the gene into messenger RNA (mRNA), transferRNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) orany other RNA product, and the translation of mRNA into polypeptide(s).If the final desired product is a biochemical, expression includes thecreation of that biochemical and any precursors. Expression of a geneproduces a “gene product.” As used herein, a gene product can be eithera nucleic acid, e.g., a messenger RNA produced by transcription of agene, or a polypeptide which is translated from a transcript. Geneproducts described herein further include nucleic acids with posttranscriptional modifications, e.g., polyadenylation, or polypeptideswith post translational modifications, e.g., methylation, glycosylation,the addition of lipids, association with other protein subunits,proteolytic cleavage, and the like.

As used herein, the term “sample” refers to any biological materialobtained from a subject or patient. In one aspect, a sample can compriseblood, peritoneal fluid, CSF, saliva or urine. In other aspects, asample can comprise whole blood, blood plasma, blood serum, B cellsenriched from blood samples, and cultured cells (e.g., B cells from asubject). A sample can also include a biopsy or tissue sample includingneural tissue. In still other aspects, a sample can comprise whole cellsand/or a lysate of the cells. Blood samples can be collected by methodsknown in the art. In one aspect, the pellet can be resuspended byvortexing at 4° C. in 200 μl buffer (20 mM Tris, pH. 7.5, 0.5% Nonidet,1 mM EDTA, 1 mM PMSF, 0.1M NaCl, IX Sigma Protease Inhibitor, and IXSigma Phosphatase Inhibitors 1 and 2). The suspension can be kept on icefor 20 minutes with intermittent vortexing. After spinning at 15,000×gfor 5 minutes at about 4° C., aliquots of supernatant can be stored atabout −70° C.

Diseases:

Unless stated otherwise, the terms “disorder” and “disease” are usedinterchangeably herein and comprise any undesired physiological changein a subject, an animal, an isolated organ, tissue or cell/cell culture.

In T2D genetic determinants and environmental factors lead to thedevelopment of insulin resistance followed by a compensatory increase inbeta-cell mass and insulin and amylin (IAPP) secretion to maintainnormal blood glucose levels. The resulting high concentrations of amylinfavor the formation of toxic hIAPP oligomers and deposition of hIAPPfibrils which is found in more than 90% of T2D patients. The depositionof hIAPP correlates with the reduction in insulin producing beta-cellsand has also been proposed to play a role for the loss of β-cells inpancreatic islets transplanted into individuals with T1D. The presentapplication provides several human-derived antibodies from pools ofhealthy donors or obese donors with high risk for T2D but absence ofdisease, which were cloned and produced recombinantly as describedherein below in more detail.

However, in one embodiment of the present invention the antibodies ofthe present invention, binding molecules having substantially the samebinding specificities of any one thereof, the polynucleotides, thevectors or the cells of the present invention are used for thepreparation of a pharmaceutical or diagnostic composition forprophylactic and therapeutic treatment, monitoring the progression or aresponse to treatment and/or diagnosis of diseases from the group ofDiabetes mellitus diseases, comprising type 1 diabetes (T1D),gestational diabetes, pre-diabetes, latent autoimmune diabetes of adults(LADA; type 1,5 diabetes) and/or type 2 diabetes (T2D).

In a preferred embodiment the antibodies of the present invention,binding molecules having substantially the same binding specificities ofany one thereof, the polynucleotides, the vectors or the cells of thepresent invention are used for the preparation of a pharmaceutical ordiagnostic composition for prophylactic and therapeutic treatment,monitoring the progression or a response to treatment and/or diagnosisof a group of disorders generally characterized by symptoms such asmetabolic changes preceding, causing, and/or connected/associated withor linked to T2D comprising diseases that cause damage to the pancreasand could therefore lead to diabetes comprising chronic pancreatitis,cystic fibrosis, pancreatic cancer; in diseases that increase the riskof T2D comprising Alzheimer's disease, Huntington's disease; and/or incardiovascular diseases linked or not with obesity and T2D. In onepreferred embodiment, symptoms generally characterizing theabove-mentioned diseases comprise disturbed insulin sensitivity andincreased secretion of insulin and/or hIAPP in a subject.

In one embodiment, the above-mentioned specific symptoms associatedgroup of disorders comprises gestational diabetes, pre-diabetes (whenhigh blood glycaemia is not reaching the T2D threshold or insulinresistance); metabolic syndrome in general as a risk factor fordeveloping diabetes or as a condition that could exist prior diabetes;Islet amyloidosis in general as a risk factor for developing diabetes oras a condition that could exist prior diabetes; obesity in general as arisk factor for developing diabetes or as a condition that could existprior diabetes and/or beta-cell failure following clinical pancreaticislet transplantation.

Furthermore, the antibodies of the present invention, binding moleculeshaving substantially the same binding specificities of any one thereof,the polynucleotides, the vectors or the cells of the present inventionare used for the preparation of a composition for detection of achanged, i.e. increased or decreased secretion of amylin as compared toamylin secretion in a healthy subject in differential diagnostic of type1 diabetes, latent autoimmune diabetes of adults (LADA, Type 1,5diabetes) in comparison to T2D forms, or in disorders preceding an overtT2D, such as gestational diabetes or pre-diabetes; in diseases thatcause damage to the pancreas and could therefore lead to diabetes suchas chronic pancreatitis, cystic fibrosis, pancreatic cancer; in diseasesthat increase the risk of T2D such as Alzheimer's disease, Huntington'sdisease; and/or in cardiovascular diseases linked or not with obesityand T2D

Disorders such as obesity and insulin resistance/hyperinsulinemia areobserved often as a predisposition and/or as a symptom of T2D which canlead to elevated circulating levels of islet amyloid polypeptide (IAPP)already ahead the overt form of T2D. Amylin (hIAPP) oligomers, fibrils,and plaques have been found accumulating not only within pancreas andkidneys but as well within the heart in patients with obesity andinsulin resistance. This accumulation has been observed in connection ofan altered cellular Ca²⁺ homeostasis which in turn may contribute tocardiac dysfunction in such patients.

Therefore, in one embodiment the antibodies of the present invention,binding molecules having substantially the same binding specificities ofany one thereof, the polynucleotides, the vectors or the cells of thepresent invention are used for the preparation of a pharmaceutical ordiagnostic composition for prophylactic and therapeutic treatment,amelioration, monitoring the progression or a response to treatmentand/or for diagnosis of a group of disorders following to T2D orresulting from the metabolic changes preceding and/or causing T2D, i.e.the metabolic changes occurring in the pre-diabetic state, wherein thegroup of disorders comprises heart disease, strokes, diabeticretinopathy, kidney failure, renal failure, ketoacidosis and nonketotichyperosmolar coma.

More than 20 neurodegenerative disorders (see, e.g., Table 1 on page 511in M. Ristow, J. Mol. Med 82 (2004), 510-529) are known to be associatedwith diabetes mellitus, increased insulin resistance and obesity,disturbed insulin sensitivity, and excessive or impaired insulinsecretion. Therefore, in one embodiment of the present invention theantibodies of the present invention, binding molecules havingsubstantially the same binding specificities of any one thereof, thepolynucleotides, the vectors or the cells of the present invention areused for the preparation of a pharmaceutical or diagnostic compositionfor prophylactic and therapeutic treatment, monitoring the progressionor a response to treatment and/or diagnosis of neurodegenerativedisorders comprising Alzheimer disease (AD), ataxia-telangiectasia (AT),Bardet-Biedl syndrome (BBS), Friedreich ataxia (FRDA), Huntingtondisease, myotonic dystrophy, narcolepsy, Parkinson disease, Prader-Willisyndrome, and Werner syndrome.

The impaired glucose tolerance scarcely induces complications beingcharacteristic for diabetes mellitus, but has a higher risk of the onsetof diabetes mellitus than normal-type, which may be a cause formacrovascular diseases.

The “insulin resistance” means conditions of reduced sensitivity toinsulin. Insulin has been known to exhibit a wider range of effects suchas, in addition to an effect on the glucose metabolism, an effect onlipid metabolism or effects on the blood vessel and the kidney. Once thesensitivity to insulin is reduced, not only glucose metabolicabnormality but also lipid metabolic abnormality such ashypertriglycemia, decreased HDL plasma level, or hypertension as anabnormality of insulin effects on the blood vessel or the kidney may beinduced.

As used herein, the term “diabetic” in a human generally and currentlymeans a random plasma or blood glucose concentration of ≥200 mg/dL(≥11.1 mmol/L) or a fasting plasma glucose ≥126 mg/dL (≥7.0 mmol/L) or a2 hour post-load glucose ≥200 mg/dL (≥11.1 mmol/L) during an oralglucose tolerance test. In addition or alternatively the term “diabetic”is used for subjects showing one or more of the clinical symptoms ofdiabetes including increased thirst (polydipsia), frequent urination(polyuria), extreme hunger, unexplained weight loss, fatigue and blurryvision, vulnerability to slow-healing sores and frequent infection,including those of the bladder, vagina and gums and/or areas of darkenedskin (acanthosis nigricans).

As used herein, the term “non-diabetic” in a human generally andcurrently means a fasting plasma glucose level of <100 mg/dL (5.6mmol/dL) or a 2 hour post-load glucose<140 mg/dL (<7.8 mmol/dL) duringan oral glucose tolerance test.

As used herein, the term “pre-diabetic” in a human generally andcurrently means a fasting plasma glucose level of 100-125 mg/dL (5.6-6.9mmol/L) or a 2 hour post-load glucose 140-199 mg/L (7.8-11.1 mmol/L)during an oral glucose tolerance test. Unless stated otherwise, theterms “pre-diabetic,” “prodromal” and “presymptomatic” are usedinterchangeably herein and describing the pre-clinical phase of T2D.

In addition or alternatively levels of glycosylated hemoglobin (HbA1c)may be used in diagnosing diabetes in a subject. At elevated HbA1clevels at or beyond the threshold of 6.5% a diagnosis of diabetes ismade, i.e. the term “diabetic” is generally and currently used in ahuman, while levels from 5.7% to 6.4% point to high risk for developingboth diabetes and cardiovascular disease and are a marker of“pre-diabetes,” or a “pre-diabetic/presymptomatic” state in a human. Theterm “non-diabetic” in a human generally and currently means then HbA1clevels below the threshold of 5.7%.

Rodent Models of Type II Diabetes Mellitus in Drug Discovery

Examples of conventionally reported model animals which spontaneouslydevelop Type II diabetes include KK-Ay mice (Nishimura M., Exp. Anim 18,147-157, 1969), NSY mice (Ueda H., et al., Diabetologia 38, 503-508,1995), db/db mice (Hummel K. P., et al., Science 153, 1127-1128, 1966),ob/ob mice (Herberg L. & Kley H K, Horm. Metab. Res. 7, 410-5, 1975),and AKITA mice (Yoshioka M., et al., Diabetes 46, 887-894, 1997).

Of these animals, KK-Ay mice and NSY mice are models with obesity anddb/db mice and ob/ob mice are models with obesity due to an abnormalityin leptin receptors or in leptin production. On the other hand, AKITAmice are a model for diabetes caused by an abnormality in pancreatic βcells.

As a non-obese Type II diabetes model animal, for example, a model mouseis so far reported in Japanese Patent Application No. 2004-65181. Thismouse exhibits abnormal insulin secretion.

However, the antibodies of the present invention are preferably testedand characterized in transgenic animals, e.g., rodents expressing hIAPPsuch as rats transgenic for human amylin in the pancreatic β-cells (HIPrats) as described in Butler et al., Diabetes 53 (2004), 1509-1516 andin Matveyenko and Butler, Diabetes 55 (2006), 2106-2114. Morepreferably, type 2 diabetes mouse models overexpressing human IAPP areused as described in Matveyenko and Butler (2006), ILAR J. 47(3):225-233, and summarized in Table 2 on page 228 therein. Because of thehIAPP-overexpression such model animals validly display T2D-symptomssuch as the hyperglycamic state. In general, the term “hyperglycemia” or“hyperglycemic state” refers to significantly increased fasting plasmaglucose levels on two consecutive measurements taken at different timeintervals and when compared to non-transgenic control littermates.Absolute values measured in hyperglycemic animals might depend from theparticular animal model used, e.g., in h-IAPP (hemizygous)/ob/+ mice,≥˜15-20 mM glucose; in h-IAPP (homozygous)/FVB/N mice ≥˜11 mM glucose.

Methods for studying diabetes include measurement of physiologicalchanges and analysis of blood or plasma of diabetic in comparison tohealthy, non-diabetic animals. These measurements include, but are notlimited to, growth dynamics, body mass index (BMI), lean mass index(LMI), food and water intake, sex differences, fasting and random bloodglucose, triglycerides (TG), lipoproteins, cholesterol, liver weight andliver lipids, kidney size and function, a glucose tolerance test (GTT),insulin tolerance test (ITT), blood insulin concentration, pancreaticislet cell morphology, high-fat diets, and caloric restriction.

As used herein, the terms “random” and “non-fasting” generally means atany time during the day or night without regard to time since the lastmeal.

As used herein, the term “fasting” generally means no caloric intake forat least 12 hours.

It is appreciated that these definitions are the currently acceptedguidelines practitioners generally follow according to the AmericanDiabetes Association (ADA) and the German Diabetes Association (GDA).Guidelines may change over time and vary by region or country and dependupon the group or institution (e.g. ADA, World Health Organization,NIDDK/NIH, CDC, GDA etc.) providing the guidelines, known to thoseskilled in the art. Physicians may also use clinical experience, thepatient's past medical history, and/or other information when decidingon a diagnosis and treatment. These definitions may therefore changeover time according to advances in science and medicine.

Treatment:

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development of diabetes.Beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which themanifestation of the condition or disorder is to be prevented.

If not stated otherwise the term “drug,” “medicine,” or “medicament” areused interchangeably herein and shall include but are not limited to all(A) articles, medicines and preparations for internal or external use,and any substance or mixture of substances intended to be used fordiagnosis, cure, mitigation, treatment, or prevention of disease ofeither man or other animals; and (B) articles, medicines andpreparations (other than food) intended to affect the structure or anyfunction of the body of man or other animals; and (C) articles intendedfor use as a component of any article specified in clause (A) and (B).The term “drug,” “medicine,” or “medicament” shall include the completeformula of the preparation intended for use in either man or otheranimals containing one or more “agents,” “compounds”, “substances” or“(chemical) compositions” as and in some other context also otherpharmaceutically inactive excipients as fillers, disintegrants,lubricants, glidants, binders or ensuring easy transport,disintegration, disaggregation, dissolution and biological availabilityof the “drug,” “medicine,” or “medicament” at an intended targetlocation within the body of man or other animals, e.g., at the skin, inthe stomach or the intestine. The terms “agent,” “compound”, or“substance” are used interchangeably herein and shall include, in a moreparticular context, but are not limited to all pharmacologically activeagents, i.e. agents that induce a desired biological or pharmacologicaleffect or are investigated or tested for the capability of inducing sucha possible pharmacological effect by the methods of the presentinvention.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, e.g., a humanpatient, for whom diagnosis, prognosis, prevention, or therapy isdesired.

Pharmaceutical Carriers:

Pharmaceutically acceptable carriers and administration routes can betaken from corresponding literature known to the person skilled in theart. The pharmaceutical compositions of the present invention can beformulated according to methods well known in the art; see for exampleRemington: The Science and Practice of Pharmacy (2000) by the Universityof Sciences in Philadelphia, ISBN 0-683-306472, Vaccine Protocols. 2ndEdition by Robinson et al., Humana Press, Totowa, N.J., USA, 2003;Banga, Therapeutic Peptides and Proteins: Formulation, Processing, andDelivery Systems. 2nd Edition by Taylor and Francis (2006), ISBN:0-8493-1630-8. Examples of suitable pharmaceutical carriers are wellknown in the art and include phosphate buffered saline solutions, water,emulsions, such as oil/water emulsions, various types of wetting agents,sterile solutions etc. Compositions comprising such carriers can beformulated by well-known conventional methods. These pharmaceuticalcompositions can be administered to the subject at a suitable dose.Administration of the suitable compositions may be effected by differentways. Examples include administering a composition containing apharmaceutically acceptable carrier via oral, intranasal, rectal,topical, intraperitoneal, intravenous, intramuscular, subcutaneous,subdermal, transdermal, intrathecal, and intracranial methods. Aerosolformulations such as nasal spray formulations include purified aqueousor other solutions of the active agent with preservative agents andisotonic agents. Such formulations are preferably adjusted to a pH andisotonic state compatible with the nasal mucous membranes.Pharmaceutical compositions for oral administration, such as singledomain antibody molecules (e.g., “Nanobodies™”) etc. are also envisagedin the present invention. Such oral formulations may be in tablet,capsule, powder, liquid or semi-solid form. A tablet may comprise asolid carrier, such as gelatin or an adjuvant. Formulations for rectalor vaginal administration may be presented as a suppository with asuitable carrier; see also O'Hagan et al., Nature Reviews, DrugDiscovery 2(9) (2003), 727-735. Further guidance regarding formulationsthat are suitable for various types of administration can be found inRemington's Pharmaceutical Sciences, Mace Publishing Company,Philadelphia, Pa., 17th ed. (1985) and corresponding updates. For abrief review of methods for drug delivery see Langer, Science 249(1990), 1527-1533.

II. Antibodies of the Present Invention

The present invention generally relates to human anti-IAPP antibodiesand antigen-binding fragments thereof, which preferably demonstrate theimmunological binding characteristics and/or biological properties asoutlined for the antibodies illustrated in the Examples. In accordancewith the present invention human monoclonal antibodies specific for IAPPand/or proIAPP were cloned from a pool of healthy human subjects.

In the course of the experiments performed in accordance with thepresent invention antibodies in conditioned media of human memory B cellcultures were screened in parallel for binding to aggregated oligomeric,fibrillar and/or non-fibrillar IAPP and/or proIAPP protein—and bovineserum albumin (BSA). Only B-cell cultures positive for aggregated IAPPand/or proIAPP protein but not for BSA were subjected to antibodycloning.

Due to this measure, several antibodies could be isolated. Selectedantibodies were further analyzed for class and light chain subclassdetermination. Selected relevant antibody messages from memory B cellcultures are then transcribed by RT-PCR, cloned and combined intoexpression vectors for recombinant production; see the appendedExamples. Recombinant expression of the human antibodies in HEK293 orCHO cells and the subsequent characterization of their bindingspecificities towards human IAPP and/or proIAPP protein (FIGS. 3 and 4;Example 1), and their distinctive binding to pathologically aggregatedforms thereof (FIG. 5; Example 2) confirmed that for the first timehuman antibodies have been cloned that are highly specific for IAPPand/or proIAPP protein and distinctively recognize the pathologicallyaggregated forms of IAPP and/or proIAPP protein, such as IAPP fibrils. Asecond round of experiments confirmed the above findings as shown inFIGS. 7-9 and in Example 4. Furthermore, mouse chimeric antibodiesgenerated according to the present invention and comprising CDR domainsof the human antibodies of the present invention have shown equalbinding affinity to human IAPP as the human antibodies as shown in FIGS.11 and 12 and in Example 6.

Thus, the present invention generally relates to an isolated naturallyoccurring human monoclonal anti-islet amyloid polypeptide (IAPP)antibody and binding fragments, derivatives and variants thereof. In oneembodiment of the invention, the antibody is capable of binding humanIAPP and/or proIAPP.

In one embodiment, the present invention is directed to an anti-IAPPand/or anti-proIAPP antibody, or antigen-binding fragment, variant orderivatives thereof, where the antibody specifically binds to the sameepitope of IAPP as a reference antibody selected from the groupconsisting of NI-203.9A2, NI-203.19H8, NI-203.26C11, NI-203.8E3,NI-203.11B12, NI-203.205F8, NI-203.9B3, NI-203.19F2, and NI-203.15C7.Epitope mapping identified a sequence within the human IAPP including aa19-SSNNFGA-25 (SEQ ID NO: 4) as the unique linear epitope recognized byantibody NI-203.19H8 of this invention, a sequence within the human IAPPincluding aa 2-CNTATCA-8 (SEQ ID NO: 5) as the unique linear epitoperecognized by antibody NI-203.26C11 of this invention, and a sequencewithin the human IAPP including aa 10-QRLANFLVHS-19 (SEQ ID NO: 71) asthe unique linear epitope recognized by antibody NI-203.15C7 of thisinvention (see FIGS. 5A and 5B and Example 3). Therefore, in oneembodiment the antibody of the present invention is provided, whereinthe antibody specifically binds an IAPP epitope which comprises theamino acid sequence SSNNFGA (SEQ ID NO: 4), CNTATCA (SEQ ID NO: 5) orQRLANFLVHS (SEQ ID NO: 71). As described in detail in Example 3 theepitopes of recombinant IAPP antibodies NI-203.9A2, NI-203.8E3, andNI-203.19F2 antibodies could not be identified so far, indicating thatthese antibodies probably bind nonlinear epitopes.

Furthermore, in one embodiment, the present invention is directed to ananti-IAPP and/or anti-proIAPP antibody, or antigen-binding fragment,variant or derivatives thereof, where the antibody specifically binds tothe same epitope of proIAPP as a reference antibody selected from thegroup consisting of antibodies NI-203.1D10, NI-203.2A11, NI-203.10C4,NI-203.20H9, NI-203.26D2, NI-203.60H3 and NI-203.26C11.

Furthermore, without intending to be bound by initial experimentalobservations as demonstrated in the Example 4 and shown in FIG. 7, thehuman monoclonal NI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3anti-IAPP antibodies of the present invention are preferablycharacterized in specifically binding to pathological IAPP aggregates(fibrils in this Example) and not substantially recognizing IAPP in thephysiological form in the pancreas. The same is expected to apply tohuman monoclonal NI-203.19F2 and NI-203.15C7 anti-IAPP antibodies.Hence, the present invention provides a set of human anti-IAPP and/oranti-proIAPP antibodies with binding specificities, which are thusparticularly useful for diagnostic and therapeutic purposes. Thus, inone embodiment the present invention provides antibodies, which arecapable of specifically binding pathologically aggregated forms of IAPPand/or proIAPP (see FIG. 5 and FIGS. 7-9, and Examples 2 and 4). Forfurther details and a summarizing overview in respect of bindingspecificities of the present invention see also FIGS. 7 and 8 and thedescription below.

In one embodiment, the antibody of the present invention exhibits thebinding properties of the exemplary NI-203.9A2, NI-203.19H8,NI-203.26C11, NI-203.8E3, NI-203.19F2, and NI-203.15C7 antibodies asdescribed in the Examples. In addition, or alternatively, the anti-IAPPand/or anti-proIAPP antibody of the present invention preferentiallyrecognizes pathologically aggregated anti-IAPP and/or anti-proIAPP, suchas IAPP fibrils rather than physiological IAPP and/or proIAPP monomers,in particular when analyzed according to Examples 2 to 4. In oneembodiment thus, the antibody of the present invention does notsubstantially recognize physiological IAPP. The term “does notsubstantially recognize” when used in the present application todescribe the binding affinity of an molecule of a group comprising anantibody, a fragment thereof or a binding molecule for a specific targetmolecule, antigen and/or conformation of the target molecule and/orantigen means that the molecule of the aforementioned group binds saidmolecule, antigen and/or conformation with a binding affinity which isat least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold or9-fold less than the dissociation constant (KD) of the molecule of theaforementioned group for binding another molecule, antigen and/orconformation. Preferably the term “does not substantially recognize”when used in the present application means that the molecule of theaforementioned group binds said molecule, antigen and/or conformationwith a binding affinity which is at least or 10-fold, 20-fold, 50-fold,100-fold, 1000-fold or 10000-fold less than the dissociation constant(KD) of said molecule of the aforementioned group for binding to anothermolecule, antigen and/or conformation. In addition, or alternatively,the anti-IAPP and/or anti-proIAPP antibody of the present inventionbinds to disease causing aggregated forms of human anti-IAPP and/oranti-proIAPP, in particular those described in Example 4. In thiscontext, the binding specificities may be in the range as shown for theexemplary NI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3antibodies in FIG. 4, respective FIG. 5, i.e. having half maximaleffective concentrations (EC50) of about 1 pM to 500 nM, preferably anEC50 of about 10 pM to 100 nM, most preferably an EC50 of about 100 pMto 50 nM for human IAPP as shown for NI-203.19H8, or an EC50 of about100 pM to 10 nM for human IAPP as shown for NI-203.9A2, NI-203.26C11 andNI-203.8E3. In this context, the experimental results as provided inExample 4 and shown FIG. 4, respective FIG. 5, also indicate that inaddition or alternatively some of the anti-IAPP antibodies of thepresent invention do not substantially recognize proIAPP as shown forthe exemplary antibodies NI-203.9A2, NI-203.19H8 and NI-203.8E3. In oneembodiment thus, the antibody of the present invention does notsubstantially recognize proIAPP.

In addition, or alternatively, the anti-IAPP antibody of the presentinvention binds specifically besides to mature IAPP to the precursorform of IAPP, i.e. proIAPP as well, in particular as described inExample 1. In this context, the binding specificities may be in therange as shown for the exemplary NI-203.26C11 antibody in FIG. 3,respective FIG. 4, i.e. having half maximal effective concentrations(EC50) of about 1 pM to 500 nM, preferably an EC50 of about 10 pM to 400nM, more preferably an EC50 of about 100 pM to 400 nM or an EC50 ofabout 100 pM to 300 nM, most preferably an EC50 of about 1 nM to 300 nMfor aggregated pro-IAPP as shown for NI-203.26C11.

In addition, or alternatively, the anti-IAPP antibody of the presentinvention binds specifically to mature IAPP and does not or does notsubstantially bind to the precursor form of IAPP, i.e. proIAPP, inparticular as described in Example 1. In this context, the bindingspecificities may be in the range as shown for the exemplary NI-203.9A2,NI-203.19H8 and NI-203.8E3 antibodies in FIG. 4, respective FIG. 5 andindicated above.

In one embodiment, the antibody of the present invention exhibits thebinding properties of exemplary antibodies NI-203.1D10, NI-203.2A11,NI-203.10C4, NI-203.20H9, NI-203.26D2 and NI-203.60H3 preferably bindingproIAPP over proIAPP. However, the proIAPP antibodies have been obtainedby a screening for antibodies specifically binding to the N-terminalflanking region of proIAPP which is not present in IAPP, as there issome evidence for the role of N-terminal unprocessed proIAPP rather thanthe full-length proIAPP peptide in amyloid formation and cell death.Thereby, in one embodiment the anti-proIAPP antibody of the presentinvention, such as exemplary antibodies NI-203.1D10, NI-203.2A11,NI-203.10C4, NI-203.20H9, NI-203.26D2 and NI-203.60H3 does not bindsubstantially or does not bind IAPP.

Some purified antibodies bind to a wide array of biomolecules, e.g.,proteins. As the skilled artisan will appreciate, the term specific isused herein to indicate that other biomolecules than IAPP and/or proIAPPproteins or fragments thereof do not significantly bind to theantigen-binding molecule, e.g., one of the antibodies of the presentinvention. Preferably, the level of binding to a biomolecule other thanIAPP and/or proIAPP results in a binding affinity which is at most only20% or less, 10% or less, only 5% or less, only 2% or less or only 1% orless (i.e. at least 5, 10, 20, 50 or 100 fold lower) of the affinity toIAPP and/or proIAPP, respectively; see e.g., Example 1 and FIG. 3.Furthermore, the anti-IAPP and/or anti-proIAPP antibodies of the presentinvention binding specifically to IAPP and/or proIAPP aggregates asvalidated by experiments showing that the antibodies of the presentinvention do not recognize pathological Aβ amyloid in Alzheimer'sdisease human brain and have only minimal cross-reactive bindingqualities to several protein candidates with misfolding/aggregationpropensities, as shown with the exemplary antibodies NI-203.9A2,NI-203.19H8 and NI-203.26C11 on paraffin-embedded brain sections of apatient diagnosed with Alzheimer's disease and by direct ELISAexperiments; see Example 5 and FIG. 10. Therefore, in one embodiment theantibody of the present invention is provided, which does notsubstantially recognize amyloid-β peptide (Aβ₁₋₄₂).

In one embodiment the anti-IAPP and/or anti-proIAPP antibody of thepresent invention preferably binds preferentially to aggregated forms ofIAPP and/or proIAPP, IAPP and/or proIAPP fibrils and/or oligomers; seeexperimental results by direct ELISA in Example 2 and in pancreas ofpatients diagnosed with T2D and on diabetic cat pancreases byimmunohistochemical staining described in Example 4 and shown in FIGS. 7and 9 respectively. In one embodiment the antibody of the presentinvention preferentially binds to aggregated forms of IAPP and/orproIAPP, wherein the aggregates comprise, essentially consist of orconsist of fibrillar forms of and/or fibrillar oligomers of IAPP. Inanother embodiment the antibody of the present invention preferentiallybinds to aggregated forms of IAPP and/or proIAPP, wherein the aggregatescomprise, essentially consist of or consist of non-fibrillar forms ofand/or non-fibrillar oligomers of IAPP. In yet another embodiment theantibody of the present invention preferentially binds to aggregatedforms of IAPP and/or proIAPP, wherein the aggregates comprise,essentially consist of or consist of either fibrillar and non-fibrillarforms of IAPP and/or proIAPP and/or fibrillar and non-fibrillaroligomers of IAPP and/or proIAPP. In still another embodiment theanti-IAPP and/or anti-proIAPP antibody of the present inventionpreferentially binds to both native IAPP and/or proIAPP andpathologically aggregated forms of IAPP and/or proIAPP; see experimentalresults as exemplified in Example 2 and Example 3 by direct ELISA.

As mentioned before, aggregates comprising IAPP and/or proIAPP can alsobe found associated with amyloid deposits in pancreatic islets of T2Dpatients. Therefore, in one embodiment the antibody of the presentinvention may be useful in treatment of the T2D.

The present invention is also drawn to an antibody, or antigen-bindingfragment, variant or derivatives thereof, where the antibody comprisesan antigen-binding domain identical to that of an antibody selected fromthe group consisting of NI-203.9A2, NI-203.19H8, NI-203.26C11,NI-203.8E3, NI-203.11B12, NI-203.205F8, NI-203.9B3, NI-203.19F2,NI-203.15C7, NI-203.1D10, NI-203.2A11, NI-203.10C4, NI-203.20H9,NI-203.26D2, and NI-203.60H3.

The present invention further exemplifies several such bindingmolecules, e.g., antibodies and binding fragments thereof, which may becharacterized by comprising in their variable region, e.g., bindingdomain at least one complementarity determining region (CDR) of theV_(H) and/or V_(L) variable region comprising any one of the amino acidsequences depicted in FIG. 1 or in FIG. 2. The corresponding nucleotidesequences encoding the above-identified variable regions are set forthin Table II respective Table III below. Exemplary sets of CDRs of theabove amino acid sequences of the V_(H) and/or V_(L) region are depictedin FIG. 1 and in FIG. 2. However, as discussed in the following theperson skilled in the art is well aware of the fact that in addition oralternatively CDRs may be used, which differ in their amino acidsequence from those set forth in FIG. 1 respective in FIG. 2 by one,two, three or even more amino acids in case of CDR2 and CDR3. Therefore,in one embodiment the antibody of the present invention or an IAPPand/or proIAPP binding fragment thereof is provided comprising in itsvariable region at least one complementarity determining region (CDR) asdepicted in FIG. 1 and/or one or more CDRs thereof comprising one ormore amino acid substitutions.

In one embodiment, the antibody of the present invention is any one ofthe antibodies comprising an amino acid sequence of the V_(H) and/orV_(L) region as depicted in FIG. 1 or a V_(H) and/or V_(L) regionthereof comprising one or more amino acid substitutions. Preferably, theantibody of the present invention is characterized by the preservationof the cognate pairing of the heavy and light chain as was present inthe human B-cell.

Alternatively, the antibody of the present invention is an antibody orantigen-binding fragment, derivative or variant thereof, which competesfor binding to IAPP and/or proIAPP with at least one of the antibodieshaving the V_(H) and/or V_(L) region as depicted in FIG. 1.

Experimental results provided in Example 4 suggest that some of theanti-IAPP and/or anti-proIAPP antibodies of the present inventionpreferentially bind to disease causing aggregated forms of humananti-IAPP and/or anti-proIAPP over the physiological forms of theproteins. In one embodiment thus, the antibody of the present inventionpreferentially recognizes IAPP and/or proIAPP aggregates overphysiological IAPP and/or proIAPP. Furthermore, in one embodiment, theantibody of the present invention preferentially recognizes IAPPaggregates comprising IAPP oligomers and/or fibrils over physiologicalIAPP. In another embodiment, the antibody of the present inventionpreferentially recognizes IAPP aggregates comprising non-fibrillar IAPPand/or non-fibrillar IAPP oligomers over physiological IAPP.

As already indicated before, some of the antibodies of the presentinvention have been shown to be capable of binding both, IAPP and itsprecursor form proIAPP. Furthermore, some of the antibodies of thepresent invention have been isolated from human patients because oftheir capability to bind proIAPP. Therefore, in one embodiment theantibody of the present invention is capable of binding proIAPP.

Therefore, alternatively or in addition to the above, in one embodimentthe antibody of the present invention or an IAPP and/or proIAPP bindingfragment thereof is provided comprising in its variable region at leastone complementarity determining region (CDR) as depicted in FIG. 2and/or one or more CDRs thereof comprising one or more amino acidsubstitutions.

In one embodiment, the antibody of the present invention is any one ofthe antibodies comprising an amino acid sequence of the V_(H) and/orV_(L) region as depicted in FIG. 2 or a V_(H) and/or V_(L) regionthereof comprising one or more amino acid substitutions. Preferably, theantibody of the present invention is characterized by the preservationof the cognate pairing of the heavy and light chain as was present inthe human B-cell.

Alternatively, the antibody of the present invention is an antibody orantigen-binding fragment, derivative or variant thereof, which competesfor binding to IAPP and/or proIAPP with at least one of the antibodieshaving the V_(H) and/or V_(L) region as depicted in FIG. 2.

The antibody of the present invention may be human, in particular fortherapeutic applications. Alternatively, the antibody of the presentinvention is a rodent, rodentized or chimeric rodent-human antibody,preferably a murine, murinized or chimeric murine-human antibody or arat, ratinized or chimeric rat-human antibody which are particularlyuseful for diagnostic methods and studies in animals. In one embodimentthe antibody of the present invention is a chimeric rodent-human or arodentized antibody.

Furthermore, in one embodiment, the chimeric antibody of the presentinvention exhibits the binding properties of the exemplary NI-203.9A2,NI-203.19H8 and NI-203.26C11 murine chimeric antibodies as described inthe Examples. Further, the mouse chimeric antibodies of the presentinvention bind with a high affinity to human IAPP fibrils as describedin Example 6. Preferably, the binding affinity of chimeric antibodies issimilar to their human counterparts. In this context, the bindingspecificities may be in the range as shown for the exemplary NI-203.9A2,NI-203.19H8 and NI-203.26C11 murine chimeric antibodies with an EC₅₀ of18.6 nM, 23.9 nM and 11.5 nM respectively as described in Example 6 andshown in FIG. 11 and Table C therein. No binding was observed on BSA.

In one embodiment the antibody of the present invention is provided bycultures of single or oligoclonal B-cells that are cultured and thesupernatant of the culture, which contains antibodies produced by saidB-cells, is screened for presence and affinity of anti-IAPP and/orproIAPP antibodies therein. The screening process comprises screeningfor binding to native monomeric, fibrillar or non-fibrillar aggregateslike oligomers of hIAPP derived from a synthetic full-length hIAPPpeptide of the amino acid sequence represented by SEQ ID NO: 1; and/or aseparate and independent screening for binding to a synthetic peptidederived from human proIAPP (N-terminal fragment) of the amino acidsequence TPIESHQVEKRKCNTATCATQR represented by SEQ ID NO: 7.

In addition or alternatively the screening process for presence andaffinity of anti-IAPP and/or proIAPP antibodies may comprise the stepsof a sensitive tissue amyloid plaque immunoreactivity (TAPIR) assay suchas described in international application WO2004/095031, the disclosurecontent of which is incorporated herein by reference, performed here inanalogy for amyloid deposits on pancreatic islets. Furthermore oralternatively, screens on pancreas sections for binding to anti-IAPPand/or proIAPP such as described in analogy in international applicationWO2008/081008 for brain and spinal cord sections may be performed.

As mentioned above, due to its generation upon a human immune responsethe human monoclonal antibody of the present invention will recognizeepitopes which are of particular pathological relevance and which mightnot be accessible or less immunogenic in case of immunization processesfor the generation of, for example, mouse monoclonal antibodies and invitro screening of phage display libraries, respectively. Accordingly,it is prudent to stipulate that the epitope of the human anti-IAPPand/or proIAPP antibody of the present invention is unique and no otherantibody which is capable of binding to the epitope recognized by thehuman monoclonal antibody of the present invention exists; see also FIG.5A and which show the unique epitope of antibody NI-203.19H8 respectiveantibody NI-203.26C11 of this invention. A further indication for theuniqueness of the antibodies of the present invention is the fact that,as indicated in Example 3, antibodies NI-203.9A2 and NI-203.8E3 of thepresent invention bind assumable conformational epitopes of IAPPaggregates, which as indicated above, are of particular pathologicalrelevance and might be as well not obtainable by the usual processes forantibody generation, such as immunization or in vitro libraryscreenings.

Therefore, in one embodiment the present invention also extendsgenerally to anti-IAPP antibodies and IAPP binding molecules whichcompete with the human monoclonal antibody of the present invention forspecific binding to IAPP. The present invention is more specificallydirected to an antibody, or antigen-binding fragment, variant orderivatives thereof, where the antibody specifically binds to the sameepitope of IAPP as a reference antibody selected from the groupconsisting of NI-203.9A2, NI-203.19H8, NI-203.26C11, NI-203.8E3,NI-203.19F2, and NI-203.15C7.

Furthermore, in one embodiment the present invention also extendsgenerally to anti-IAPP and/or anti-proIAPP antibodies and IAPP and/oranti-proIAPP binding molecules which compete with the human monoclonalantibody of the present invention for specific binding to proIAPP. Thepresent invention is therefore, more specifically also directed to anantibody, or antigen-binding fragment, variant or derivatives thereof,where the antibody specifically binds to the same epitope of proIAPP asa reference antibody selected from the group consisting of NI-203.1D10,NI-203.2A11, NI-203.10C4, NI-203.20H9, NI-203.26D2, NI-203.60H3 andNI-203.26C11.

In addition, or alternatively the present invention also extendsgenerally to bispecific anti-IAPP and/or anti-proIAPP antibodies andIAPP and/or anti-proIAPP binding molecules which compete with the humanmonoclonal antibody of the present invention for specific binding toboth IAPP and proIAPP. The present invention is therefore, morespecifically also directed to an antibody, or antigen-binding fragment,variant or derivatives thereof, where the antibody specifically binds tothe same epitope of IAPP and proIAPP as the exemplary antibodyNI-203.26C11. In view of the above thus, in one embodiment the presentinvention also relates to an antibody or antigen-binding molecule whichcompetes with an antibody of the present invention for specific bindingto IAPP and/or proIAPP.

Competition between antibodies is determined by an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as IAPP and/or proIAPP. Numeroustypes of competitive binding assays are known, for example: solid phasedirect or indirect radioimmunoassay (RIA), solid phase direct orindirect enzyme immunoassay (EIA), sandwich competition assay; seeStahli et al., Methods in Enzymology 9 (1983), 242-253; solid phasedirect biotin-avidin EIA; see Kirkland et al., J. Immunol. 137 (1986),3614-3619 and Cheung et al., Virology 176 (1990), 546-552; solid phasedirect labeled assay, solid phase direct labeled sandwich assay; seeHarlow and Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPress (1988); solid phase direct label RIA using I¹²⁵ label; see Morelet al., Molec. Immunol. 25 (1988), 7-15 and Moldenhauer et al., Scand.J. Immunol. 32 (1990), 77-82. Typically, such an assay involves the useof purified IAPP and/or proIAPP or aggregates, such as oligomers and/orfibrils thereof bound to a solid surface or cells bearing either ofthese, an unlabeled test immunoglobulin and a labeled referenceimmunoglobulin, i.e. the human monoclonal antibody of the presentinvention. Competitive inhibition is measured by determining the amountof label bound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Preferably, the competitive binding assay is performed under conditionsas described for the ELISA assay in the appended Examples. Antibodiesidentified by competition assay (competing antibodies) includeantibodies binding to the same epitope as the reference antibody andantibodies binding to an adjacent epitope sufficiently proximal to theepitope bound by the reference antibody for steric hindrance to occur.Usually, when a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to a common antigen by at least50% or 75%. Hence, the present invention is further drawn to anantibody, or antigen-binding fragment, variant or derivatives thereof,where the antibody competitively inhibits a reference antibody selectedfrom the group consisting of NI-203.9A2, NI-203.19H8, NI-203.26C11,NI-203.8E3, NI-203.19F2, and NI-203.15C7 from binding to IAPP.

In addition, the present invention is further drawn to an antibody, orantigen-binding fragment, variant or derivatives thereof, where theantibody competitively inhibits a reference antibody selected from thegroup consisting of NI-203.1D10, NI-203.2A11, NI-203.10C4, NI-203.20H9,NI-203.26D2, NI-203.60H3 and NI-203.26C11 from binding to proIAPP.

Further in addition or alternatively the present invention is furtherdrawn to a bispecific antibody, or antigen-binding fragment, variant orderivatives thereof, where the antibody competitively inhibits areference antibody such as the exemplary antibody NI-203.26C11 frombinding to either IAPP and proIAPP.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)), where at least oneof V_(H)-CDRs of the heavy chain variable region or at least two of theV_(H)-CDRs of the heavy chain variable region are at least 80%, 85%, 90%or 95% identical to reference heavy chain V_(H)-CDR1, V_(H)-CDR2 orV_(H)-CDR3 amino acid sequences from the antibodies disclosed herein.Alternatively, the V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 regions of theV_(H) are at least 80%, 85%, 90% or 95% identical to reference heavychain V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 amino acid sequences fromthe antibodies disclosed herein. Thus, according to this embodiment aheavy chain variable region of the invention has V_(H)-CDR1, V_(H)-CDR2and V_(H)-CDR3 polypeptide sequences related to the groups shown in FIG.1 or in FIG. 2 respectively. While FIGS. 1 and 2 show V_(H)-CDRs definedby the Kabat system, other CDR definitions, e.g., V_(H)-CDRs defined bythe Chothia system, are also included in the present invention, and canbe easily identified by a person of ordinary skill in the art using thedata presented in FIG. 1 and FIG. 2.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)) in which theV_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 groupsshown in FIG. 1 or in FIG. 2 respectively.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)) in which theV_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 groupsshown in FIG. 1 or in FIG. 2 respectively, except for one, two, three,four, five, or six amino acid substitutions in any one V_(H)-CDR. Incertain embodiments the amino acid substitutions are conservative.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (V_(L)), where at least oneof the V_(L)-CDRs of the light chain variable region or at least two ofthe V_(L)-CDRs of the light chain variable region are at least 80%, 85%,90% or 95% identical to reference light chain V_(L)-CDR1, V_(L)-CDR2 orV_(L)-CDR3 amino acid sequences from antibodies disclosed herein.Alternatively, the V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 regions of theV_(L) are at least 80%, 85%, 90% or 95% identical to reference lightchain V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 amino acid sequences fromantibodies disclosed herein. Thus, according to this embodiment a lightchain variable region of the invention has V_(L)-CDR1, V_(L)-CDR2 andV_(L)-CDR3 polypeptide sequences related to the polypeptides shown inFIG. 1 or in FIG. 2 respectively. While FIGS. 1 and 2 show V_(L)-CDRsdefined by the Kabat system, other CDR definitions, e.g., V_(L)-CDRsdefined by the Chothia system, are also included in the presentinvention.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (V_(L)) in which theV_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 groupsshown in FIG. 1 or in FIG. 2 respectively.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(L)) in which theV_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 groupsshown in FIG. 1 or in FIG. 2 respectively, except for one, two, three,four, five, or six amino acid substitutions in any one V_(L)-CDR. Incertain embodiments the amino acid substitutions are conservative.

An immunoglobulin or its encoding cDNA may be further modified. Thus, ina further embodiment the method of the present invention comprises anyone of the step(s) of producing a chimeric antibody, murinized antibody,single-chain antibody, Fab-fragment, bi-specific antibody, fusionantibody, labeled antibody or an analog of any one of those.Corresponding methods are known to the person skilled in the art and aredescribed, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”,CSH Press, Cold Spring Harbor (1988). When derivatives of saidantibodies are obtained by the phage display technique, surface plasmonresonance as employed in the BIAcore system can be used to increase theefficiency of phage antibodies which bind to the same epitope as that ofany one of the antibodies described herein (Schier, Human AntibodiesHybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995),7-13). The production of chimeric antibodies is described, for example,in international application WO89/09622. Methods for the production ofhumanized antibodies are described in, e.g., European application EP-A10 239 400 and international application WO90/07861. Further sources ofantibodies to be utilized in accordance with the present invention areso-called xenogeneic antibodies. The general principle for theproduction of xenogeneic antibodies such as human-like antibodies inmice is described in, e.g., international applications WO91/10741,WO94/02602, WO96/34096 and WO 96/33735. As discussed above, the antibodyof the invention may exist in a variety of forms besides completeantibodies; including, for example, Fv, Fab and F(ab)₂, as well as insingle chains; see e.g. international application WO88/09344. In oneembodiment therefore, the antibody of the present invention is provided,which is selected from the group consisting of a single chain Fvfragment (scFv), an F(ab′) fragment, an F(ab) fragment, and an F(ab′)₂fragment.

The antibodies of the present invention or their correspondingimmunoglobulin chain(s) can be further modified using conventionaltechniques known in the art, for example, by using amino aciddeletion(s), insertion(s), substitution(s), addition(s), and/orrecombination(s) and/or any other modification(s) known in the arteither alone or in combination. Methods for introducing suchmodifications in the DNA sequence underlying the amino acid sequence ofan immunoglobulin chain are well known to the person skilled in the art;see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold SpringHarbor Laboratory (1989) N.Y. and Ausubel, Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y. (1994). Modifications of the antibody of the invention includechemical and/or enzymatic derivatizations at one or more constituentamino acids, including side chain modifications, backbone modifications,and N- and C-terminal modifications including acetylation,hydroxylation, methylation, amidation, and the attachment ofcarbohydrate or lipid moieties, cofactors, and the like. Likewise, thepresent invention encompasses the production of chimeric proteins whichcomprise the described antibody or some fragment thereof at the aminoterminus fused to heterologous molecule such as an immunostimulatoryligand at the carboxyl terminus; see, e.g., international applicationWO00/30680 for corresponding technical details.

Additionally, the present invention encompasses peptides including thosecontaining a binding molecule as described above, for example containingthe CDR3 region of the variable region of any one of the mentionedantibodies, in particular CDR3 of the heavy chain since it hasfrequently been observed that heavy chain CDR3 (HCDR3) is the regionhaving a greater degree of variability and a predominant participationin antigen-antibody interaction. Such peptides may easily be synthesizedor produced by recombinant means to produce a binding agent usefulaccording to the invention. Such methods are well known to those ofordinary skill in the art. Peptides can be synthesized for example,using automated peptide synthesizers which are commercially available.The peptides can also be produced by recombinant techniques byincorporating the DNA expressing the peptide into an expression vectorand transforming cells with the expression vector to produce thepeptide.

Hence, the present invention relates to any binding molecule, e.g., anantibody or binding fragment thereof which is oriented towards the humananti-IAPP and/or anti-proIAPP antibodies of the present invention anddisplays the mentioned properties, i.e. which specifically recognizesIAPP and/or proIAPP. Such antibodies and binding molecules can be testedfor their binding specificity and affinity by ELISA andimmunohistochemistry as described herein, see, e.g., the Examples. Thesecharacteristics of the antibodies and binding molecules can be tested byWestern Blot as well. Preliminary results of subsequent experimentsperformed in accordance with the present invention revealed that thehuman anti-IAPP and/or anti-proIAPP antibody of the present invention,in particular antibodies NI-203.9A2, NI-203.19H8, NI-203.26C11 andNI-203.8E3 were able to differentially bind to IAPP-fibrils in ELISAtests. Furthermore, 203.9A2, NI-203.19H8 and NI-203.26C11 antibodies ofthe present invention have been shown to preferentially bind topathologies in human, such as large amyloid deposits in pancreaticislets corresponding to pathological IAPP fibrils, as visualized byThioS and Congo red staining (see FIG. 7A). The same properties areexpected to apply to antibodies NI-203.19F2 and NI-203.15C7.

Human antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 showedprominent pancreatic islet staining on amyloid-positive sections butwere not showing any staining on pancreatic islets from a T2D patientlacking amyloid deposits and from a control patient not diagnosed withT2D (see Example 4 and FIG. 7). The antibodies of the present inventionalso gave positive results on diabetic cat pancreases showing isletamyloid deposits; see FIG. 9. This binding specificity towardspathological forms of IAPP and/or proIAPP in human and animal tissueemphasizes besides the biochemical experiments showed herein (seeExamples 2 and FIG. 5) the usability of the antibodies of the presentinvention in treatment and diagnosis of diseases associated withoccurrence of aggregated IAPP and/or proIAPP in the pancreas.

As an alternative to obtaining immunoglobulins directly from the cultureof B cells or B memory cells, the cells can be used as a source ofrearranged heavy chain and light chain loci for subsequent expressionand/or genetic manipulation. Rearranged antibody genes can be reversetranscribed from appropriate mRNAs to produce cDNA. If desired, theheavy chain constant region can be exchanged for that of a differentisotype or eliminated altogether. The variable regions can be linked toencode single chain Fv regions. Multiple Fv regions can be linked toconfer binding ability to more than one target or chimeric heavy andlight chain combinations can be employed. Once the genetic material isavailable, design of analogs as described above which retain both theirability to bind the desired target is straightforward. Methods for thecloning of antibody variable regions and generation of recombinantantibodies are known to the person skilled in the art and are described,for example, Gilliland et al., Tissue Antigens 47 (1996), 1-20; Doeneckeet al., Leukemia 11 (1997), 1787-1792.

Once the appropriate genetic material is obtained and, if desired,modified to encode an analog, the coding sequences, including those thatencode, at a minimum, the variable regions of the heavy and light chain,can be inserted into expression systems contained on vectors which canbe transfected into standard recombinant host cells. A variety of suchhost cells may be used; for efficient processing, however, mammaliancells are preferred. Typical mammalian cell lines useful for thispurpose include, but are not limited to, CHO cells, HEK 293 cells, orNSO cells.

The production of the antibody or analog is then undertaken by culturingthe modified recombinant host under culture conditions appropriate forthe growth of the host cells and the expression of the coding sequences.The antibodies are then recovered by isolating them from the culture.The expression systems are preferably designed to include signalpeptides so that the resulting antibodies are secreted into the medium;however, intracellular production is also possible.

In accordance with the above, the present invention also relates to apolynucleotide encoding the antibody or equivalent binding molecule ofthe present invention, in case of the antibody preferably at least avariable region of an immunoglobulin chain of the antibody describedabove. Typically, said variable region encoded by the polynucleotidecomprises at least one complementarity determining region (CDR) of theVII and/or V_(L) of the variable region of the said antibody.

The person skilled in the art will readily appreciate that the variabledomain of the antibody having the above-described variable domain can beused for the construction of other polypeptides or antibodies of desiredspecificity and biological function. Thus, the present invention alsoencompasses polypeptides and antibodies comprising at least one CDR ofthe above-described variable domain and which advantageously havesubstantially the same or similar binding properties as the antibodydescribed in the appended examples. The person skilled in the art knowsthat binding affinity may be enhanced by making amino acid substitutionswithin the CDRs or within the hypervariable loops (Chothia and Lesk, J.Mol. Biol. 196 (1987), 901-917) which partially overlap with the CDRs asdefined by Kabat; see, e.g., Riechmann, et al, Nature 332 (1988),323-327. Thus, the present invention also relates to antibodies whereinone or more of the mentioned CDRs comprise one or more, preferably notmore than two amino acid substitutions. Preferably, the antibody of theinvention comprises in one or both of its immunoglobulin chains two orall three CDRs of the variable regions as set forth in FIG. 1 orrespectively in FIG. 2.

Binding molecules, e.g., antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention, as known by those ofordinary skill in the art, can comprise a constant region which mediatesone or more effector functions. For example, binding of the C1 componentof complement to an antibody constant region may activate the complementsystem. Activation of complement is important in the opsonization andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and may also be involved in autoimmunehypersensitivity. Further, antibodies bind to receptors on various cellsvia the Fc region, with a Fc receptor binding site on the antibody Fcregion binding to a Fc receptor (FcR) on a cell. There are a number ofFc receptors which are specific for different classes of antibody,including IgG (gamma receptors), IgE (epsilon receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of antibody to Fc receptorson cell surfaces triggers a number of important and diverse biologicalresponses including engulfment and destruction of antibody-coatedparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (called antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer and control of immunoglobulin production.

Accordingly, certain embodiments of the present invention include anantibody, or antigen-binding fragment, variant, or derivative thereof,in which at least a fraction of one or more of the constant regiondomains has been deleted or otherwise altered so as to provide desiredbiochemical characteristics such as reduced effector functions, theability to non-covalently dimerize, increased ability to localize at thesite of IAPP and/or proIAPP aggregation and deposition, reduced serumhalf-life, or increased serum half-life when compared with a whole,unaltered antibody of approximately the same immunogenicity. Forexample, certain antibodies for use in the diagnostic and treatmentmethods described herein are domain deleted antibodies which comprise apolypeptide chain similar to an immunoglobulin heavy chain, but whichlack at least a portion of one or more heavy chain domains. Forinstance, in certain antibodies, one entire domain of the constantregion of the modified antibody will be deleted, for example, all orpart of the CH2 domain will be deleted. In other embodiments, certainantibodies for use in the diagnostic and treatment methods describedherein have a constant region, e.g., an IgG heavy chain constant region,which is altered to eliminate glycosylation, referred to elsewhereherein as aglycosylated or “agly” antibodies. Such “agly” antibodies maybe prepared enzymatically as well as by engineering the consensusglycosylation site(s) in the constant region. While not being bound bytheory, it is believed that “agly” antibodies may have an improvedsafety and stability profile in vivo. Methods of producing aglycosylatedantibodies, having desired effector function are found for example ininternational application WO2005/018572, which is incorporated byreference in its entirety.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated todecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain may reduce Fc receptor binding of thecirculating modified antibody thereby increasing IAPP and/or proIAPPlocalization. In other cases it may be that constant regionmodifications consistent with the instant invention moderate complementbinding and thus reduce the serum half-life and nonspecific associationof a conjugated cytotoxin. Yet other modifications of the constantregion may be used to modify disulfide linkages or oligosaccharidemoieties that allow for enhanced localization due to increased antigenspecificity or antibody flexibility. The resulting physiologicalprofile, bioavailability and other biochemical effects of themodifications, such as IAPP and/or proIAPP localization, biodistributionand serum half-life, may easily be measured and quantified using wellknow immunological techniques without undue experimentation.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated orexchanged for alternative protein sequences to increase the cellularuptake of antibodies by way of example by enhancing receptor-mediatedendocytosis of antibodies via Fcγ receptors, LRP, or Thy1 receptors orby ‘SuperAntibody Technology’, which is said to enable antibodies to beshuttled into living cells without harming them (Expert Opin. Biol.Ther. (2005), 237-241). For example, the generation of fusion proteinsof the antibody binding region and the cognate protein ligands of cellsurface receptors or bi- or multi-specific antibodies with a specificsequences biding to IAPP and/or proIAPP as well as a cell surfacereceptor may be engineered using techniques known in the art.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated orexchanged for alternative protein sequences or the antibody may bechemically modified to increase its blood brain barrier penetration.

Modified forms of antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be made from whole precursor orparent antibodies using techniques known in the art. Exemplarytechniques are discussed in more detail herein. Antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be made or manufactured using techniques that are known inthe art. In certain embodiments, antibody molecules or fragments thereofare “recombinantly produced,” i.e., are produced using recombinant DNAtechnology. Exemplary techniques for making antibody molecules orfragments thereof are discussed in more detail elsewhere herein.

Antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention also include derivatives that are modified,e.g., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromspecifically binding to its cognate epitope. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

In particular preferred embodiments, antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention will notelicit a deleterious immune response in the animal to be treated, e.g.,in a human. In certain embodiments, binding molecules, e.g., antibodies,or antigen-binding fragments thereof of the invention are derived from apatient, e.g., a human patient, and are subsequently used in the samespecies from which they are derived, e.g., human, alleviating orminimizing the occurrence of deleterious immune responses.

De-immunization can also be used to decrease the immunogenicity of anantibody. As used herein, the term “de-immunization” includes alterationof an antibody to modify T cell epitopes; see, e.g., internationalapplications WO98/52976 and WO00/34317. For example, V_(H) and V_(L)sequences from the starting antibody are analyzed and a human T cellepitope “map” from each V region showing the location of epitopes inrelation to complementarity determining regions (CDRs) and other keyresidues within the sequence. Individual T cell epitopes from the T cellepitope map are analyzed in order to identify alternative amino acidsubstitutions with a low risk of altering activity of the finalantibody. A range of alternative V_(H) and V_(L) sequences are designedcomprising combinations of amino acid substitutions and these sequencesare subsequently incorporated into a range of binding polypeptides,e.g., IAPP and/or proIAPP-specific antibodies or immunospecificfragments thereof for use in the diagnostic and treatment methodsdisclosed herein, which are then tested for function. Typically, between12 and 24 variant antibodies are generated and tested. Complete heavyand light chain genes comprising modified V and human C regions are thencloned into expression vectors and the subsequent plasmids introducedinto cell lines for the production of whole antibody. The antibodies arethen compared in appropriate biochemical and biological assays, and theoptimal variant is identified.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.(1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas Elsevier, N.Y., 563-681 (1981), said references incorporatedby reference in their entireties. The term “monoclonal antibody” as usedherein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced. Thus, the term“monoclonal antibody” is not limited to antibodies produced throughhybridoma technology. In certain embodiments, antibodies of the presentinvention are derived from human B cells which have been immortalizedvia transformation with Epstein-Barr virus, as described herein.

In the well-known hybridoma process (Kohler et al., Nature 256 (1975),495) the relatively short-lived, or mortal, lymphocytes from a mammal,e.g., B cells derived from a human subject as described herein, arefused with an immortal tumor cell line (e.g., a myeloma cell line),thus, producing hybrid cells or “hybridomas” which are both immortal andcapable of producing the genetically coded antibody of the B cell. Theresulting hybrids are segregated into single genetic strains byselection, dilution, and re-growth with each individual straincomprising specific genes for the formation of a single antibody. Theyproduce antibodies, which are homogeneous against a desired antigen and,in reference to their pure genetic parentage, are termed “monoclonal”.

Hybridoma cells thus prepared are seeded and grown in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. Those skilledin the art will appreciate that reagents, cell lines and media for theformation, selection and growth of hybridomas are commercially availablefrom a number of sources and standardized protocols are wellestablished. Generally, culture medium in which the hybridoma cells aregrowing is assayed for production of monoclonal antibodies against thedesired antigen. The binding specificity of the monoclonal antibodiesproduced by hybridoma cells is determined by in vitro assays such asimmunoprecipitation, radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA) as described herein. After hybridoma cellsare identified that produce antibodies of the desired specificity,affinity and/or activity, the clones may be subcloned by limitingdilution procedures and grown by standard methods; see, e.g., Goding,Monoclonal Antibodies: Principles and Practice, Academic Press, pp.59-103 (1986). It will further be appreciated that the monoclonalantibodies secreted by the subclones may be separated from culturemedium, ascites fluid or serum by conventional purification proceduressuch as, for example, protein-A, hydroxylapatite chromatography, gelelectrophoresis, dialysis or affinity chromatography.

In another embodiment, lymphocytes can be selected by micromanipulationand the variable genes isolated. For example, peripheral bloodmononuclear cells can be isolated from an immunized or naturally immunemammal, e.g., a human, and cultured for about 7 days in vitro. Thecultures can be screened for specific IgGs that meet the screeningcriteria. Cells from positive wells can be isolated. IndividualIg-producing B cells can be isolated by FACS or by identifying them in acomplement-mediated hemolytic plaque assay. Ig-producing B cells can bemicromanipulated into a tube and the V_(H) and V_(L) genes can beamplified using, e.g., RT-PCR. The V_(H) and V_(L) genes can be clonedinto an antibody expression vector and transfected into cells (e.g.,eukaryotic or prokaryotic cells) for expression.

Alternatively, antibody-producing cell lines may be selected andcultured using techniques well known to the skilled artisan. Suchtechniques are described in a variety of laboratory manuals and primarypublications. In this respect, techniques suitable for use in theinvention as described below are described in Current Protocols inImmunology, Coligan et al., Eds., Green Publishing Associates andWiley-Interscience, John Wiley and Sons, New York (1991) which is hereinincorporated by reference in its entirety, including supplements.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments may be producedrecombinantly or by proteolytic cleavage of immunoglobulin molecules,using enzymes such as papain (to produce Fab fragments) or pepsin (toproduce F(ab′)2 fragments). F(ab′)2 fragments contain the variableregion, the light chain constant region and the CH1 domain of the heavychain. Such fragments are sufficient for use, for example, inimmunodiagnostic procedures involving coupling the immunospecificportions of immunoglobulins to detecting reagents such as radioisotopes.

Human antibodies, such as described herein, are particularly desirablefor therapeutic use in human patients. Human antibodies of the presentinvention are isolated, e.g., from healthy human subjects who because oftheir overweight or obesity may be suspected to be at risk of developinga metabolic disorder, e.g., T2D, or a patient with the disorder but withan unusually stable disease course or unusually mild form of thedisease. However, the healthy human subject suspected to be at risk todevelop a metabolic disorder, e.g., T2D from whom antibodies, such asdescribed herein may be isolated, may as well be selected on the basisof the presence of other risks known to enhance the chance of a personto develop a metabolic disorder, e.g., T2D. Said risks may be deducedfrom an examination of the person for risk factors associated with thedevelopment of a metabolic disorder, e.g., T2D, such as age 45 or older;overweight or obesity; close family relatives with diabetes; familybackground is African American, Alaska Native, American Indian, AsianAmerican, Hispanic/Latino, Pacific Islander American, Asian or Arabic;history of gestational diabetes; giving birth to at least one babyweighing more than 4.5 kg; blood pressure of 140/90 or higher;cholesterol levels higher than normal, with, e.g., High-densitylipoprotein (HDL) level below 40 mg/dL (equivalent to below 1 mmol/L),or triglyceride level above 200-499 mg/dL (equivalent to above 2.3-5.6mmol/L); sedentary lifestyle; diagnosis of polycystic ovary syndrome(PCOS); diagnosis of prediabetes on previous testing—an A1C (also calledHbA1c or glycohaemoglobin) level of 5.7 to 6.4 percent, impaired fastingglucose (IFG), or impaired glucose tolerance (IGT); diagnosis of otherclinical conditions associated with insulin resistance, such as aacanthosis nigricans; history of cardiovascular disease.

In case obesity or overweight of a person is used as an indicator of aperson to develop a metabolic disease, e.g., T2D, though it is prudentto expect that obese healthy and symptom-free subjects, respectively,more regularly will have developed protective anti-IAPP and/oranti-proIAPP antibodies than subjects who are diagnosed with a lessrisk, e.g., because they are not classified as obese but as overweight,or even as persons of a normal weight, subjects belonging to the lattertwo classifications may be used as well as source for obtaining a humanantibody of the present invention as well.

A subject may be classified as having normal weight, being overweight orobese based on measurements of the subjects height and weight, andcalculating the subjects Body Mass Index by the following calculation:BMI=weight [kg]/(height[m])². Based on the result, the subjects areclassified as of normal weight (BMI 18.5-24.9 kg/m2), overweight(25.0-29.9 kg/m2), or obese (≥30 kg/m2) based on current World HealthOrganization criteria (World Health Organization (2000) “Obesity:preventing and managing the global epidemic. Report of a WHOconsultation.” World Health Organ Tech Rep Ser 894: 1.253).Alternatively or in addition, the waist circumference (WC) of a subjectmay be measured, and used based on sex-specific cut-offs to define WC asnormal (<94 cm [<34.6 inches] in men and <80 cm [31.5 inches] in women),moderately increased (94-102 cm [34.6-40 inches] in men and 80-88 cm[31.5-35 inches] in women), or large (≥102 cm [≥40 inches] in menand >88 cm [≥35 inches] in women) as described in InterAct Consortium,Langenberg et al., PLoS Med. 2012 June; 9(6): e1001230, wherein ahealthy subject with a high waist circumference (large WC) moreregularly will have developed protective anti-IAPP and/or anti-proIAPPantibodies comparable with the classification as obese the highest riskto develop T2D and may be used preferably for the isolation of theseantibodies, but persons with a moderately increased or normal WC may beused for the isolation of anti-IAPP and/or anti-proIAPP antibodies aswell.

In one embodiment, an antibody of the invention comprises at least oneheavy or light chain CDR of an antibody molecule. In another embodiment,an antibody of the invention comprises at least two CDRs from one ormore antibody molecules. In another embodiment, an antibody of theinvention comprises at least three CDRs from one or more antibodymolecules. In another embodiment, an antibody of the invention comprisesat least four CDRs from one or more antibody molecules. In anotherembodiment, an antibody of the invention comprises at least five CDRsfrom one or more antibody molecules. In another embodiment, an antibodyof the invention comprises at least six CDRs from one or more antibodymolecules. Exemplary antibody molecules comprising at least one CDR thatcan be included in the subject antibodies are described herein.

Antibodies of the present invention can be produced by any method knownin the art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably by recombinant expression techniques asdescribed herein.

In one embodiment, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention comprises a synthetic constantregion wherein one or more domains are partially or entirely deleted(“domain-deleted antibodies”). In certain embodiments compatiblemodified antibodies will comprise domain deleted constructs or variantswherein the entire CH2 domain has been removed (ΔCH2 constructs). Forother embodiments a short connecting peptide may be substituted for thedeleted domain to provide flexibility and freedom of movement for thevariable region. Those skilled in the art will appreciate that suchconstructs are particularly preferred due to the regulatory propertiesof the CH2 domain on the catabolic rate of the antibody. Domain deletedconstructs can be derived using a vector encoding an IgG₁ human constantdomain, see, e.g., international applications WO02/060955 andWO02/096948A2. This vector is engineered to delete the CH2 domain andprovide a synthetic vector expressing a domain deleted IgG₁ constantregion.

In certain embodiments, antibodies, or antigen-binding fragments,variants, or derivatives thereof of the present invention areminibodies. Minibodies can be made using methods described in the art,see, e.g., U.S. Pat. No. 5,837,821 or international applicationWO94/09817.

In one embodiment, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention comprises an immunoglobulin heavychain having deletion or substitution of a few or even a single aminoacid as long as it permits association between the monomeric subunits.For example, the mutation of a single amino acid in selected areas ofthe CH2 domain may be enough to substantially reduce Fc binding andthereby increase IAPP and/or proIAPP localization. Similarly, it may bedesirable to simply delete that part of one or more constant regiondomains that control the effector function (e.g. complement binding) tobe modulated. Such partial deletions of the constant regions may improveselected characteristics of the antibody (serum half-life) while leavingother desirable functions associated with the subject constant regiondomain intact. Moreover, as alluded to above, the constant regions ofthe disclosed antibodies may be synthetic through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it may be possible to disrupt theactivity provided by a conserved binding site (e.g. Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Yet other embodiments comprise the addition ofone or more amino acids to the constant region to enhance desirablecharacteristics such as an effector function or provide for morecytotoxin or carbohydrate attachment. In such embodiments it may bedesirable to insert or replicate specific sequences derived fromselected constant region domains.

The present invention also provides antibodies that comprise, consistessentially of, or consist of, variants (including derivatives) ofantibody molecules (e.g., the V_(H) regions and/or V_(L) regions)described herein, which antibodies or fragments thereofimmunospecifically bind to IAPP and/or proIAPP. Standard techniquesknown to those of skill in the art can be used to introduce mutations inthe nucleotide sequence encoding an antibody, including, but not limitedto, site-directed mutagenesis and PCR-mediated mutagenesis which resultin amino acid substitutions. Preferably, the variants (includingderivatives) encode less than 50 amino acid substitutions, less than 40amino acid substitutions, less than 30 amino acid substitutions, lessthan 25 amino acid substitutions, less than 20 amino acid substitutions,less than 15 amino acid substitutions, less than 10 amino acidsubstitutions, less than 5 amino acid substitutions, less than 4 aminoacid substitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions relative to the reference V_(H) region,V_(H)-CDR1, V_(H)-CDR2, V_(H)-CDR3, V_(L) region, V_(L)-CDR1,V_(L)-CDR2, or V_(L)-CDR3. A “conservative amino acid substitution” isone in which the amino acid residue is replaced with an amino acidresidue having a side chain with a similar charge. Families of aminoacid residues having side chains with similar charges have been definedin the art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g., theability to bind IAPP and/or proIAPP).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, e.g., have no, orlittle, effect on an antibody's ability to bind antigen, indeed somesuch mutations do not alter the amino acid sequence whatsoever. Thesetypes of mutations may be useful to optimize codon usage, or improve ahybridoma's antibody production. Codon-optimized coding regions encodingantibodies of the present invention are disclosed elsewhere herein.Alternatively, non-neutral mis sense mutations may alter an antibody'sability to bind antigen. The location of most silent and neutralmissense mutations is likely to be in the framework regions, while thelocation of most non-neutral missense mutations is likely to be in CDR,though this is not an absolute requirement. One of skill in the artwould be able to design and test mutant molecules with desiredproperties such as no alteration in antigen-binding activity oralteration in binding activity (e.g., improvements in antigen-bindingactivity or change in antibody specificity). Following mutagenesis, theencoded protein may routinely be expressed and the functional and/orbiological activity of the encoded protein, (e.g., ability toimmunospecifically bind at least one epitope of IAPP and/or proIAPP) canbe determined using techniques described herein or by routinelymodifying techniques known in the art.

III. Polynucleotides Encoding Antibodies

A polynucleotide encoding an antibody, or antigen-binding fragment,variant, or derivative thereof can be composed of any polyribonucleotideor polydeoxribonucleotide, which may be unmodified RNA or DNA ormodified RNA or DNA. For example, a polynucleotide encoding an antibody,or antigen-binding fragment, variant, or derivative thereof can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, a polynucleotide encoding an antibody, orantigen-binding fragment, variant, or derivative thereof can be composedof triple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide encoding an antibody, or antigen-binding fragment,variant, or derivative thereof may also contain one or more modifiedbases or DNA or RNA backbones modified for stability or for otherreasons. “Modified” bases include, for example, tritylated bases andunusual bases such as inosine. A variety of modifications can be made toDNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically,or metabolically modified forms.

An isolated polynucleotide encoding a non-natural variant of apolypeptide derived from an immunoglobulin (e.g., an immunoglobulinheavy chain portion or light chain portion) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore non-essential amino acid residues.

As is well known, RNA may be isolated from the original B cells,hybridoma cells or from other transformed cells by standard techniques,such as a guanidinium isothiocyanate extraction and precipitationfollowed by centrifugation or chromatography. Where desirable, mRNA maybe isolated from total RNA by standard techniques such as chromatographyon oligo dT cellulose. Suitable techniques are familiar in the art. Inone embodiment, cDNAs that encode the light and the heavy chains of theantibody may be made, either simultaneously or separately, using reversetranscriptase and DNA polymerase in accordance with well-known methods.PCR may be initiated by consensus constant region primers or by morespecific primers based on the published heavy and light chain DNA andamino acid sequences. As discussed above, PCR also may be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase the libraries may be screened by consensus primers or largerhomologous probes, such as human constant region probes.

DNA, typically plasmid DNA, may be isolated from the cells usingtechniques known in the art, restriction mapped and sequenced inaccordance with standard, well known techniques set forth in detail,e.g., in the foregoing references relating to recombinant DNAtechniques. Of course, the DNA may be synthetic according to the presentinvention at any point during the isolation process or subsequentanalysis.

In this context, the present invention also relates to a polynucleotideencoding at least the binding domain or variable region of animmunoglobulin chain of the antibody of the present invention. In oneembodiment, the present invention provides an isolated polynucleotidecomprising, consisting essentially of, or consisting of a nucleic acidencoding an immunoglobulin heavy chain variable region (V_(H)), where atleast one of the CDRs of the heavy chain variable region or at least twoof the V_(H)-CDRs of the heavy chain variable region are at least 80%,85%, 90% or 95% identical to reference heavy chain V_(H)-CDR1,V_(H)-CDR2, or V_(H)-CDR3 amino acid sequences from the antibodiesdisclosed herein. Alternatively, the V_(H)-CDR1, V_(H)-CDR2, orV_(H)-CDR3 regions of the V_(H) are at least 80%, 85%, 90% or 95%identical to reference heavy chain V_(H)-CDR1, V_(H)-CDR2, andV_(H)-CDR3 amino acid sequences from the antibodies disclosed herein.Thus, according to this embodiment a heavy chain variable region of theinvention has V_(H)-CDR1, V_(H)-CDR2, or V_(H)-CDR3 polypeptidesequences related to the polypeptide sequences shown in FIG. 1, orrespectively has V_(H)-CDR1, V_(H)-CDR2, or V_(H)-CDR3 polypeptidesequences related to the polypeptide sequences shown in FIG. 2.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain variable region(V_(L)), where at least one of the V_(L)-CDRs of the light chainvariable region or at least two of the V_(L)-CDRs of the light chainvariable region are at least 80%, 85%, 90% or 95% identical to referencelight chain V_(L)-CDR1, V_(L)-CDR2, or V_(L)-CDR3 amino acid sequencesfrom the antibodies disclosed herein. Alternatively, the V_(L)-CDR1,V_(L)-CDR2, or V_(L)-CDR3 regions of the V_(L) are at least 80%, 85%,90% or 95% identical to reference light chain V_(L)-CDR1, V_(L)-CDR2,and V_(L)-CDR3 amino acid sequences from the antibodies disclosedherein. Thus, according to this embodiment a light chain variable regionof the invention has V_(L)-CDR1, V_(L)-CDR2, or V_(L)-CDR3 polypeptidesequences related to the polypeptide sequences shown in FIG. 1, orrespectively has V_(L)-CDR1, V_(L)-CDR2, or V_(L)-CDR3 polypeptidesequences related to the polypeptide sequences shown in FIG. 2.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region(V_(H)) in which the V_(H)-CDR1, V_(H)-CDR2, and V_(H)-CDR3 regions havepolypeptide sequences which are identical to the V_(H)-CDR1, V_(H)-CDR2,and V_(H)-CDR3 groups shown in FIG. 1 or respectively are identical tothe V_(H)-CDR1, V_(H)-CDR2, and V_(H)-CDR3 groups as shown in FIG. 2.

As known in the art, “sequence identity” between two polypeptides or twopolynucleotides is determined by comparing the amino acid or nucleicacid sequence of one polypeptide or polynucleotide to the sequence of asecond polypeptide or polynucleotide. When discussed herein, whether anyparticular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can bedetermined using methods and computer programs/software known in the artsuch as, but not limited to, the BESTFIT program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).BESTFIT uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2 (1981), 482-489, to find the bestsegment of homology between two sequences. When using BESTFIT or anyother sequence alignment program to determine whether a particularsequence is, for example, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference polypeptide sequence and that gaps in homology of up to5% of the total number of amino acids in the reference sequence areallowed.

In a preferred embodiment of the present invention, the polynucleotidecomprises, consists essentially of, or consists of a nucleic acid havinga polynucleotide sequence of the V_(H) or V_(L) region of an anti-IAPPand/or anti-proIAPP antibody as depicted in Table II or in Table III. Inthis respect, the person skilled in the art will readily appreciate thatthe polynucleotides encoding at least the variable domain of the lightand/or heavy chain may encode the variable domain of both immunoglobulinchains or only one. In one embodiment therefore, the polynucleotidecomprises, consists essentially of, or consists of a nucleic acid havinga polynucleotide sequence of the V_(H) and the V_(L) region of ananti-IAPP and/or anti-proIAPP antibody as depicted in Table II or thesequence of the V_(H) and the V_(L) region of an anti-IAPP and/oranti-proIAPP antibody as depicted in Table III.

TABLE IINucleotide sequences of the V_(H) and V_(L) region of IAPP antibodies.Nucleotide sequences of variable heavy (VH) and  Antibodyvariable light (VL/VK) chains NI-203.9A2-V_(H)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGGCTCTCCTGTGCAGCCTCTGGATTCACGTTTAGCACCTTTGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAGTGGTAGTGGTGATAATACATACTATGCAGACTCCCTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACACTATATCTGCAAGTGAACAGCCTGAGACCCGAGGACACGGCCGTTTATTACTGTGCGAAAAGTCCCTCGTCACTTCTGGCCACCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 11 NI-203.9A2-V_(K)GAAATTGTGTTGACACAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTGAGAGTATTAATAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGGCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGCACAATAGTTATTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 13 NI-203.19H8-V_(H)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGACGTCCCTGAGACTCTCCTGTGCAGCGTCTGGGTTCACCTTCAGCAGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGCCTGGAGTGGGTGGCAATTATATGGTATGATGGAAGTAAGGAATATTATGCAGACTCCCTGAAGGGCCGAGTCACCATCTCCAGAGACAATTCCGAGAACACTCTCTATCTGCAACTGCACACCCTGAGAGTCGAGGACACGGCTGTGTATTTCTGTGCGAGGACAATCGCATCGGCCACCGTGGACCACGGTATGGACGTCTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 15 NI-203.19H8-V_(K)GATGTTGTGATGACTCAGTCTCCTTCGTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCACGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAGACCAGGGAAAGCCCCTAACCTCCTGATCTTTGGAGCATCGAGGTTGCAAAGTGGGGTCTCACCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGACTAACAATTTCCCTCCCACCTTCGGCCAAGGGACACGACTGGAGATTAAA SEQ ID NO: 17 N1-203.26C11-V_(H)CAGGTGCAGCTGCAGGAGTCGGGCCCAGGATTGGTGAAGCCTTCTCAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTAATTACTACTGGACCTGGATCCGGCAGCCCGCCGGGAAGGGACTGGAGTGGATTGGGCATATCTATTCCAGTGGGACCACCAATTACAACCCCTCCCTCGAGAGTCGAGTCACCATTTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAGCCTGAACTCTGTGACCGCCGCAGACACGGCCGTTTATTACTGTGCGAGACCACTGGCTACAGTTCCGGATGCTTTTAATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCG SEQ ID NO: 19 NI-203.26C11-V_(K)GAAATTGTGATGACTCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAAGTGCAAGTCCAGCCAGAGTGTTTTATACAGCAATAAGAACTTCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCATTTACTGGGCATCTACTCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAGTATTATAGTAATCCTAACACTTTTGGCCAGGGGACCAAGGTGGAGATCAAA SEQ ID NO: 21NI-203.8E3-V_(H) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAACCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGTAGTCACACTATCAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATGGGAGGGATCATCCCCATCTTTGGTACAGCAAACTACGCACAGAAGTTTCAGGACAGAGTCACGGTTACCGCGGACAAATCCACGAATACAGCCTACATGGAGTTGAGTAGCCTCAGACCTGAGGACACGGCCGTGTATTACTGTGCGAAGGGGGAACTGGAACCACGAATCCTCTACTACTACGGTATGGACGTCTGGGGCCGAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 23 NI-203.8E3-V_(K)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTATACAGTGATGGAAACACCTACTTGAATTGGTTTCACCAGAGGCCAGGCCAATCTCCAAGGCGGCTAATTTATAAGGTTTCTAATCGTGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTTCAAATTGGCCAGGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 25NI-203.11B12-V_(H) CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAATGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAACTACTATTTACACTGGGTGCGACAGGCCCCTGGACAAGGACTTGAGTGGATGGGAATAATCAACCCTAGTGCTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAACTGAGCAGCCTGAAATCTGAAGACACGGCCGTCTATTACTGTGCGAGAGATTCCGCTGGGATACAGATATGGTTCAGGGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCG SEQ ID NO: 27 NI-203.11B12-V_(H)CAGCCTGTGCTGACTCAGCCACCCTCTGCCTCTGCTTCCCTGGGATCCTCGGTCAAGCTCACCTGCACTCTGAACAGTGGGCACAGTAGCTACACCATCGCATGGCATCAGCAGCAGCCAGGGAAGGCCCCTCGGTACTTGATGAAGGTTGAACATAATGGAAACTACAACAAGGGGAGCGGACTTCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGACCGCTACCTCGCCATCTCCAACCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGAGACCTGGGACACTAGCACTAGGGTCTTCGGCGGAGGGACCAAGCTGACCGTCCTA SEQ ID NO: 29NI-203.205F8-V_(H) CAGGTGCAGCTGCAGGAGTCGGGCCCCGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGACTCCGTCAGCAGTGGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTATATTCCTGTGCGAGAGTCCCCTATGGTTACGGATATAGGGGCTACGATGGGGCTTGGTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 31NI-203.205F8-V_(K) GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACCGGTTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA SEQ ID NO:33 NI-203.9B3-V_(H)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCGTCAGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATCTGGTATGATGGAACTAAGAAGTACTATGCAGACTCCGTGAAGGGCCGATTCACCACCTCCAGAGACAATTCCAAGAATACGCTGTCTCTGCAAATGAACAGCCTGAGAGCCGAGGACTCGGCTGTGTATTACTGTGCGAGAGGCTTTAGCAGCAGCTGGGAGTTTGGGTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 35 NI-203.9B3-V_(L)CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCGGTTACATTTATGGTTATAACTATGTCTCCTGGTACCAACAGCACCCCGGCAAAGCCCCCAAAGTCATGATTTATGAGGTCACTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGGCTCCAGGCTGAGGATGAGGCTGTTTATTACTGCGCCTCATATGCAGGCAGCAACAATGTAGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA SEQ ID NO: 37NI-203.19F2-V_(H) GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAGGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCAACTTCTTGAGCTATTCCATCAGTTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCGATCTTTGGTACACCAAACTACGCACAGAAGTTCCAAGGAAGAGTCACAATTACGGCGGACAAATCGACGAGGACAGCCTACATGGAGCTGAGCAGCCTGAGATTTGATGACACGGCCGTCTATTATTGTGCGGATGCGACAAGACCGGGTACAGCAGCCTCTGGTTTCTATTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 63NI-203.19F2-V_(K) GAAATTGTGATGACACAGTCTCCAGACACCCTGTCTGTGTCTCCAGGTGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAACAACAACTTAGCCTGGTTCCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATTCCAGCCAGATTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTACAGTCTGAAGATTTTGCAGTTTATTTCTGTCAGCAGAGTCACAATTGGCCCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA SEQ ID NO: 65 NI-203.15C7-V_(H)GAGGTGCAGCTGGTGGAGACTGGGGGAGGCGTGGTCCAGCCTGGGATGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTACCTATACTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGTCATTTATATCATATGATGGAAGGGATAAATACTACGCAGATTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACATGTTGTATCTGCAAATGAACAGCCTGAGAGATGAGGACATGGCTGTGTATTACTGTGCGACTCTGCAAGTATGGCAACTCTACGATTACTACGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 67 NI-203.15C7-V_(L)CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCTTGGTATCAGCAACTCCCAGGAACAGCCCCCAAACTCCTCATTTATAACAGTGATAAGCGACCCTCAGGGATTCCTGACCGATTCTCTGCCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGGCTCCAGACTGGGGACGAGGCCGATTATTACTGCGCAACATGGGATACCAGACTGAGTGCTGGGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTT SEQ ID NO: 69

TABLE IIINucleotide sequences of the VH and VL region of proIAPP antibodies.Nucleotide sequences of variable heavy (VH) and Antibodyvariable light (VL/VK) chains NI-203.1D10-V_(H)GAGGTGCAGCTGGTGCAGTCTGGCGCAGAAGTGAAGAAGCCCGGGGAGTCTCTCAGAATCTCCTGTAAGGCTTCTGGATACAGCTTCACCAACTCTTGGATCGCCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGACTACGTGGGTATCATCTATCCTGGTGACTCTGATACCAAGTATGGCCCGTCCTTCCAAGGCCACGTCACTATCTCAGCCGACAACTTCGCCAACACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCCGACACCGCCATCTATTATTGTGCGAGACGGGCAGCAGCGGCTATTAACTGGTTCGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 39 NI-203.1D10-V_(K)GACATCCAGTTGACCCAGTCTCCACTCTCCCTGTCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGCCAGAGCCTCCTGCATCCTAATGGAAACGACTATTTGGATTGGTACGTGCAGAAGCCAGGGCAGTCTCCACAGATCGTGATCTACATGGGTTCTAATCGGGCCGCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGACTTATTACTGCCTGCAAGCTCTACGCGGGTACACTTTTGGCCAGGGGACCAAGGTGGAAATCAAA SEQ ID NO: 41 NI-203.2A11-V_(H)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCGTCTGGATTCACCTTCAGCAGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTGTACGGTATGATGGAAGTAATAAGTACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACTCGCTGTCTCTTCAAATGAACAGTCTGAGAACTGAAGACACGGCTGTATATTACTGCGCGAAAGAACAGGAGGACCACAAGGAAGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 43 NI-203.2A11-V_(K)GAAATTGTGATGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGAGTTACCACCATAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGATATTCCCGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGTCTGCAGTCTGAAGACTTTGCAGTTTATTACTGTCAGCAGTATAACCAGTGGCCCCTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAA SEQ ID NO: 45 NI-203.10C4-V_(H)GAGGTGCAGCTGGTGGAGTCTGGGGCTGAAGTGAGGAAGCCTGGGGCCTCAGTGAGGGTCTCCTGCCAGACATCTGGATACAGCGTCACCGACTACTATCTACACTGGGTGCGACAGGCCCCTGGACAGGGCCTTGAGTGGATGGGAGTGATGAACCCGAGCAATGGAAACGTGGGCTACCCACAGAAGTTTCAGGGCCGAGTCACCATGACCGCAGACACGTCCACGGGCACAGTGTACATGGTGTTGACCGGCCTTACGGCTGGGGACACGGCCGTCTACTACTGTGCCAGAGGCGGGTCCACGCCGGGTCAGGAAGTAAGGAGTCCCCACGTCCTTGACCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 47 NI-203.10C4-V_(K)GATGTTGTGATGACTCAGTCTCCCCTCTCTCTGTCCGTCACCCCTGGACAGCCGGCCTCCATCTCCTGCAGGTCTGATGAGAGCCTCCTGCATAGTGATGGAAGGACCTATTTGTATTGGTATCTACAGAAGCCCGGCCAGCCTCCTCAGCTCCTGATCTATGAAGTTTCCAACCGGTTCTCGGGAGTGCCAAATAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTGAAAATCAGCCGGGTGGAGGCTGAGGATGTTGGCGTTTATTACTGCATGCAGGGTGTACACTTTCCTCAGACGTTCGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 49NI-203.20H9-V_(H) CAGGTGCAGCTGGTGCAATCTGGGTCTGAGTTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACATCTTCAGTAAACATGGTATCAACTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATAGGATGGATCAACACCAATACGGGGAACCCAACATATGCCCAGGACTTCACAGGACGATTTGTCTTCTCCTTGGACACCTCTGTCAGCACGGCATATCTGGAGATCAGCAGCCTAAAGGCTGAGGACACTGCCGTGTATTACTGTGCGAGAGAATCAGAGCCGATTTTTGGAGTTATCTATTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 51 NI-203.20H9-V_(K)GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGCGTCACCATCACTTGCCGGGCAAGCCAGAGCATAAGCACTAATTTAAATTGGTATCAGAAGAAACCAGGACAAGCCCCTACGGTCTTGATCTATGCTGCGTCCAGTTTGCAAGGTGGGGTCCCATCAAGGTTCAGGGGCCGGGGATCTGGGACATATTTCACTCTCACCATCAGCGGTCTTCAACCTGAAGATTTTGCAACTTATTACTGTCAACACAATTACAATGATTTGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 53 NI-203.26D2-V_(H)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGGTTCACGTTCAGAACCTGTGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAATGGGTGGCATTTGTTCGGTCTGATGGAACTACTAGATATTACGCAGACTCCCTGATGGGCCGCTTCACCATCTCCAGAGACAATTCCAAGAACTCGCTGTATCTTCAAATGAACAGTCTGAGACCTGAGGACACGGCTCTTTATTACTGTGCGAGGGAAAAGGAGGATCACAGGGAAGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 55 NI-203.26D2-V_(K)GAAATTGTGATGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGCGTGTTAGCACTGTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCACCAGGGCCACTGATATCCCCGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCACTCTGCAATCTGAAGACTCTGCAGTTTATTACTGTCAGCAGTATAATAGGTGGCCCCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 57 NI-203.60H3-V_(H)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGCACGCCCTGGAGGCTCCCTGAGACTCTCCTGTGCAGTCGCTGGATTCACTTTCAGTGGTTATGAAATGAATTGGGTCCGCCAGGCACCAGGGAAGGGGCTGGAGTGGATTTCATATATTAGCGGTCCTGGGGATGTGATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTTTCTACAGATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTATTGTACGAGAGTCCCCCCTGACATCAGCTATGGATTTGATTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 59 NI-203.60H3-V_(K)GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTACGAGACAGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCCATGATACAGACATTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCACTGGATCTGGGACAGATTTCACTCTCACCATCAGCGGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCTACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 61

The present invention also includes fragments of the polynucleotides ofthe invention, as described elsewhere. Additionally polynucleotideswhich encode fusion polynucleotides, Fab fragments, and otherderivatives, as described herein, are also contemplated by theinvention.

The polynucleotides may be produced or manufactured by any method knownin the art. For example, if the nucleotide sequence of the antibody isknown, a polynucleotide encoding the antibody may be assembled fromchemically synthesized oligonucleotides, e.g., as described in Kutmeieret al., BioTechniques 17 (1994), 242, which, briefly, involves thesynthesis of overlapping oligonucleotides containing portions of thesequence encoding the antibody, annealing and ligating of thoseoligonucleotides, and then amplification of the ligated oligonucleotidesby PCR.

Alternatively, a polynucleotide encoding an antibody, or antigen-bindingfragment, variant, or derivative thereof may be generated from nucleicacid from a suitable source. If a clone containing a nucleic acidencoding a particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the antibody may bechemically synthesized or obtained from a suitable source (e.g., anantibody cDNA library, or a cDNA library generated from, or nucleicacid, preferably polyA⁺ RNA, isolated from, any tissue or cellsexpressing the IAPP and/or proIAPP-specific antibody, such as hybridomacells selected to express an antibody) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody, or antigen-binding fragment, variant, or derivativethereof is determined, its nucleotide sequence may be manipulated usingmethods well known in the art for the manipulation of nucleotidesequences, e.g., recombinant DNA techniques, site directed mutagenesis,PCR, etc. (see, for example, the techniques described in Sambrook etal., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel et al., eds.,Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998),which are both incorporated by reference herein in their entireties), togenerate antibodies having a different amino acid sequence, for exampleto create amino acid substitutions, deletions, and/or insertions.

IV. Expression of Antibody Polypeptides

Following manipulation of the isolated genetic material to provideantibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention, the polynucleotides encoding the antibodiesare typically inserted in an expression vector for introduction intohost cells that may be used to produce the desired quantity of antibody.Recombinant expression of an antibody, or fragment, derivative or analogthereof, e.g., a heavy or light chain of an antibody which binds to atarget molecule is described herein. Once a polynucleotide encoding anantibody molecule or a heavy or light chain of an antibody, or portionthereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablelinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g.,international applications WO 86/05807 and WO 89/01036; and U.S. Pat.No. 5,122,464) and the variable domain of the antibody may be clonedinto such a vector for expression of the entire heavy or light chain.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present invention as a vehicle forintroducing into and expressing a desired gene in a host cell. As knownto those skilled in the art, such vectors may easily be selected fromthe group consisting of plasmids, phages, viruses and retroviruses. Ingeneral, vectors compatible with the instant invention will comprise aselection marker, appropriate restriction sites to facilitate cloning ofthe desired gene and the ability to enter and/or replicate in eukaryoticor prokaryotic cells. For the purposes of this invention, numerousexpression vector systems may be employed. For example, one class ofvector utilizes DNA elements which are derived from animal viruses suchas bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus,baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Othersinvolve the use of polycistronic systems with internal ribosome bindingsites. Additionally, cells which have integrated the DNA into theirchromosomes may be selected by introducing one or more markers whichallow selection of transfected host cells. The marker may provide forprototrophy to an auxotrophic host, biocide resistance (e.g.,antibiotics) or resistance to heavy metals such as copper. Theselectable marker gene can either be directly linked to the DNAsequences to be expressed, or introduced into the same cell byco-transformation. Additional elements may also be needed for optimalsynthesis of mRNA. These elements may include signal sequences, splicesignals, as well as transcriptional promoters, enhancers, andtermination signals.

In particularly preferred embodiments the cloned variable region genesare inserted into an expression vector along with the heavy and lightchain constant region genes (preferably human) as discussed above. Inone embodiment, this is accomplished using a proprietary expressionvector of Biogen IDEC, Inc., referred to as NEOSPLA, and disclosed inU.S. Pat. No. 6,159,730. This vector contains the cytomegaloviruspromoter/enhancer, the mouse beta globin major promoter, the SV40 originof replication, the bovine growth hormone polyadenylation sequence,neomycin phosphotransferase exon 1 and exon 2, the dihydrofolatereductase gene and leader sequence. This vector has been found to resultin very high level expression of antibodies upon incorporation ofvariable and constant region genes, transfection in CHO cells, followedby selection in G418 containing medium and methotrexate amplification.Of course, any expression vector which is capable of elicitingexpression in eukaryotic cells may be used in the present invention.Examples of suitable vectors include, but are not limited to plasmidspcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2,pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (available fromInvitrogen, San Diego, Calif.), and plasmid pCI (available from Promega,Madison, Wis.). In general, screening large numbers of transformed cellsfor those which express suitably high levels if immunoglobulin heavy andlight chains is routine experimentation which can be carried out, forexample, by robotic systems. Vector systems are also taught in U.S. Pat.Nos. 5,736,137 and 5,658,570, each of which is incorporated by referencein its entirety herein. This system provides for high expression levels,e.g., >30 pg/cell/day. Other exemplary vector systems are disclosede.g., in U.S. Pat. No. 6,413,777.

In other preferred embodiments the antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention may beexpressed using polycistronic constructs such as those disclosed in USpatent application publication no. 2003-0157641 A1 and incorporatedherein in its entirety. In these expression systems, multiple geneproducts of interest such as heavy and light chains of antibodies may beproduced from a single polycistronic construct. These systemsadvantageously use an internal ribosome entry site (IRES) to providerelatively high levels of antibodies. Compatible IRES sequences aredisclosed in U.S. Pat. No. 6,193,980 which is also incorporated herein.Those skilled in the art will appreciate that such expression systemsmay be used to effectively produce the full range of antibodiesdisclosed in the instant application. Therefore, in one embodiment thepresent invention provides a vector comprising the polynucleotideencoding at least the binding domain or variable region of animmunoglobulin chain of the antibody, optionally in combination with apolynucleotide that encodes the variable region of the otherimmunoglobulin chain of said binding molecule.

More generally, once the vector or DNA sequence encoding a monomericsubunit of the antibody has been prepared, the expression vector may beintroduced into an appropriate host cell. Introduction of the plasmidinto the host cell can be accomplished by various techniques well knownto those of skill in the art. These include, but are not limited to,transfection including lipotransfection using, e.g., Fugene® orlipofectamine, protoplast fusion, calcium phosphate precipitation, cellfusion with enveloped DNA, microinjection, and infection with intactvirus. Typically, plasmid introduction into the host is via standardcalcium phosphate co-precipitation method. The host cells harboring theexpression construct are grown under conditions appropriate to theproduction of the light chains and heavy chains, and assayed for heavyand/or light chain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the invention includes host cells comprising apolynucleotide encoding an antibody of the invention, or a heavy orlight chain thereof, or at least the binding domain or variable regionof an immunoglobulin thereof, which preferably are operable linked to aheterologous promoter. In addition or alternatively the invention alsoincludes host cells comprising a vector, as defined hereinabove,comprising a polynucleotide encoding at least the binding domain orvariable region of an immunoglobulin chain of the antibody, optionallyin combination with a polynucleotide that encodes the variable region ofthe other immunoglobulin chain of said binding molecule. In preferredembodiments for the expression of double-chained antibodies, a singlevector or vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain is advantageouslyplaced before the heavy chain to avoid an excess of toxic free heavychain; see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad.Sci. USA 77 (1980), 2197. The coding sequences for the heavy and lightchains may comprise cDNA or genomic DNA.

As used herein, “host cells” refers to cells which harbor vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene. In descriptions of processes for isolation ofantibodies from recombinant hosts, the terms “cell” and “cell culture”are used interchangeably to denote the source of antibody unless it isclearly specified otherwise. In other words, recovery of polypeptidefrom the “cells” may mean either from spun down whole cells, or from thecell culture containing both the medium and the suspended cells.

A variety of host-expression vector systems may be utilized to expressantibody molecules for use in the methods described herein. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing antibody coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, NSO, BLK, 293, 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter). Preferably, bacterial cells such asEscherichia coli, and more preferably, eukaryotic cells, especially forthe expression of whole recombinant antibody molecule, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese Hamster Ovary (CHO) cells, in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus is an effective expression system for antibodies;see, e.g., Foecking et al., Gene 45 (1986), 101; Cockett et al.,Bio/Technology 8 (1990), 2.

The host cell line used for protein expression is often of mammalianorigin; those skilled in the art are credited with ability topreferentially determine particular host cell lines which are bestsuited for the desired gene product to be expressed therein. Exemplaryhost cell lines include, but are not limited to, CHO (Chinese HamsterOvary), DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA(human cervical carcinoma), CVI (monkey kidney line), COS (a derivativeof CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK,WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast),HAK (hamster kidney line), SP2/O (mouse myeloma), P3x63-Ag3.653 (mousemyeloma), BFA-1c1BPT (bovine endothelial cells), RAH (human lymphocyte)and 293 (human kidney). CHO and 293 cells are particularly preferred.Host cell lines are typically available from commercial services, theAmerican Tissue Culture Collection or from published literature.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which stably express theantibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11(1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska& Szybalski, Proc. Natl. Acad. Sci. USA 48 (1992), 202), and adeninephosphoribosyltransferase (Lowy et al., Cell 22 (1980), 817) genes canbe employed in tk-, hgprt- or aprt-cells, respectively. Also,anti-metabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77 (1980), 357; O'Hare et al., Proc. Natl.Acad. Sci. USA 78 (1981), 1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78(1981), 2072); neo, which confers resistance to the aminoglycoside G-418Goldspiel et al., Clinical Pharmacy 12 (1993), 488-505; Wu and Wu,Biotherapy 3 (1991), 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32(1993), 573-596; Mulligan, Science 260 (1993), 926-932; and Morgan andAnderson, Ann. Rev. Biochem. 62 (1993), 191-217; TIB TECH 11 (1993),155-215; and hygro, which confers resistance to hygromycin (Santerre etal., Gene 30 (1984), 147. Methods commonly known in the art ofrecombinant DNA technology which can be used are described in Ausubel etal. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, N Y (1990); and in Chapters 12 and 13, Dracopoli et al.(eds), Current Protocols in Human Genetics, John Wiley & Sons, N Y(1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification, for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Academic Press, New York, Vol. 3.(1987). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase; see Crouse et al., Mol. Cell. Biol. 3(1983), 257. In vitro production allows scale-up to give large amountsof the desired polypeptides. Techniques for mammalian cell cultivationunder tissue culture conditions are known in the art and includehomogeneous suspension culture, e.g. in an airlift reactor or in acontinuous stirrer reactor, or immobilized or entrapped cell culture,e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-) affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.

Genes encoding antibodies, or antigen-binding fragments, variants orderivatives thereof of the invention can also be expressed innon-mammalian cells such as bacteria or insect or yeast or plant cells.Bacteria which readily take up nucleic acids include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the heterologous polypeptides typically becomepart of inclusion bodies. The heterologous polypeptides must beisolated, purified and then assembled into functional molecules. Wheretetravalent forms of antibodies are desired, the subunits will thenself-assemble into tetravalent antibodies; see, e.g., internationalapplication WO02/096948.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2 (1983),1791), in which the antibody coding sequence may be ligated individuallyinto the vector in frame with the lacZ coding region so that a fusionprotein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13(1985), 3101-3109; Van Heeke & Schuster, J. Biol. Chem. 24 (1989),5503-5509); and the like. pGEX vectors may also be used to expressforeign polypeptides as fusion proteins with glutathione S-transferase(GST). In general, such fusion proteins are soluble and can easily bepurified from lysed cells by adsorption and binding to a matrix ofglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available, e.g., Pichia pastoris. For expression inSaccharomyces, the plasmid YRp7, for example, (Stinchcomb et al., Nature282 (1979), 39; Kingsman et al., Gene 7 (1979), 141; Tschemper et al.,Gene 10 (1980), 157) is commonly used. This plasmid already contains theTRP1 gene which provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example ATCC No. 44076 orPEP4-1 (Jones, Genetics 85 (1977), 12). The presence of the trpl lesionas a characteristic of the yeast host cell genome then provides aneffective environment for detecting transformation by growth in theabsence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencemay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Once an antibody molecule of the invention has been recombinantlyexpressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention,can be purified according to standard procedures of the art, includingfor example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,e g ammonium sulfate precipitation, or by any other standard techniquefor the purification of proteins; see, e.g., Scopes, “ProteinPurification”, Springer Verlag, N.Y. (1982). Alternatively, a preferredmethod for increasing the affinity of antibodies of the invention isdisclosed in US patent publication 2002-0123057 A1. In one embodimenttherefore, the present invention also provides a method for preparing ananti-IAPP or an anti-proIAPP antibody or immunoglobulin chain(s)thereof, said method comprising:

-   (a) culturing the host cell as defined hereinabove, which cell    comprised a polynucleotide or a vector as defined hereinbefore; and-   (b) isolating said antibody or immunoglobulin chain(s) thereof from    the culture.

Furthermore, in one embodiment the present invention also relates to anantibody or immunoglobulin chain(s) thereof encoded by a polynucleotideas defined hereinabove or obtainable by said method for preparing ananti-IAPP or an anti-proIAPP antibody or immunoglobulin chain(s)thereof.

V. Fusion Proteins and Conjugates

In certain embodiments, the antibody polypeptide comprises an amino acidsequence or one or more moieties not normally associated with anantibody. Exemplary modifications are described in more detail below.For example, a single-chain Fv antibody fragment of the invention maycomprise a flexible linker sequence, or may be modified to add afunctional moiety (e.g., PEG, a drug, a toxin, or a label such as afluorescent, radioactive, enzyme, nuclear magnetic, heavy metal and thelike)

An antibody polypeptide of the invention may comprise, consistessentially of, or consist of a fusion protein. Fusion proteins arechimeric molecules which comprise, for example, an immunoglobulin IAPPand/or proIAPP-binding domain with at least one target binding site, andat least one heterologous portion, i.e., a portion with which it is notnaturally linked in nature. The amino acid sequences may normally existin separate proteins that are brought together in the fusion polypeptideor they may normally exist in the same protein but are placed in a newarrangement in the fusion polypeptide. Fusion proteins may be created,for example, by chemical synthesis, or by creating and translating apolynucleotide in which the peptide regions are encoded in the desiredrelationship.

The term “heterologous” as applied to a polynucleotide or a polypeptide,means that the polynucleotide or polypeptide is derived from a distinctentity from that of the rest of the entity to which it is beingcompared. For instance, as used herein, a “heterologous polypeptide” tobe fused to an antibody, or an antigen-binding fragment, variant, oranalog thereof is derived from a non-immunoglobulin polypeptide of thesame species, or an immunoglobulin or non-immunoglobulin polypeptide ofa different species.

As discussed in more detail elsewhere herein, antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention may further be recombinantly fused to a heterologouspolypeptide at the N- or C-terminus or chemically conjugated (includingcovalent and non-covalent conjugations) to polypeptides or othercompositions. For example, antibodies may be recombinantly fused orconjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs,radionuclides, or toxins; see, e.g., international applicationsWO92/08495; WO91/14438; WO89/12624; U.S. Pat. No. 5,314,995; andEuropean patent application EP 0 396 387.

Antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention can be composed of amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain amino acids other than the 20 gene-encodedamino acids. Antibodies may be modified by natural processes, such asposttranslational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature. Modifications can occur anywhere in theantibody, including the peptide backbone, the amino acid side-chains andthe amino or carboxyl termini, or on moieties such as carbohydrates. Itwill be appreciated that the same type of modification may be present inthe same or varying degrees at several sites in a given antibody. Also,a given antibody may contain many types of modifications. Antibodies maybe branched, for example, as a result of ubiquitination, and they may becyclic, with or without branching. Cyclic, branched, and branched cyclicantibodies may result from posttranslational natural processes or may bemade by synthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphatidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination;see, e.g., Proteins—Structure And Molecular Properties, T. E. Creighton,W. H. Freeman and Company, New York 2nd Ed., (1993); PosttranslationalCovalent Modification Of Proteins, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182 (1990),626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).

The present invention also provides for fusion proteins comprising anantibody, or antigen-binding fragment, variant, or derivative thereof,and a heterologous polypeptide. In one embodiment, a fusion protein ofthe invention comprises, consists essentially of, or consists of, apolypeptide having the amino acid sequence of any one or more of theV_(H) regions of an antibody of the invention or the amino acid sequenceof any one or more of the V_(L) regions of an antibody of the inventionor fragments or variants thereof, and a heterologous polypeptidesequence. In another embodiment, a fusion protein for use in thediagnostic and treatment methods disclosed herein comprises, consistsessentially of, or consists of a polypeptide having the amino acidsequence of any one, two, three of the V_(H)-CDRs of an antibody, orfragments, variants, or derivatives thereof, or the amino acid sequenceof any one, two, three of the V_(L)-CDRs of an antibody, or fragments,variants, or derivatives thereof, and a heterologous polypeptidesequence. In one embodiment, the fusion protein comprises a polypeptidehaving the amino acid sequence of a V_(H)-CDR3 of an antibody of thepresent invention, or fragment, derivative, or variant thereof, and aheterologous polypeptide sequence, which fusion protein specificallybinds to IAPP and/or proIAPP. In another embodiment, a fusion proteincomprises a polypeptide having the amino acid sequence of at least oneV_(H) region of an antibody of the invention and the amino acid sequenceof at least one V_(L) region of an antibody of the invention orfragments, derivatives or variants thereof, and a heterologouspolypeptide sequence. Preferably, the V_(H) and V_(L) regions of thefusion protein correspond to a single source antibody (or scFv or Fabfragment) which specifically binds IAPP and/or proIAPP. In yet anotherembodiment, a fusion protein for use in the diagnostic and treatmentmethods disclosed herein comprises a polypeptide having the amino acidsequence of any one, two, three or more of the V_(H) CDRs of an antibodyand the amino acid sequence of any one, two, three or more of the V_(L)CDRs of an antibody, or fragments or variants thereof, and aheterologous polypeptide sequence. Preferably, two, three, four, five,six, or more of the V_(H)-CDR(s) or V_(L)-CDR(s) correspond to singlesource antibody (or scFv or Fab fragment) of the invention. Nucleic acidmolecules encoding these fusion proteins are also encompassed by theinvention.

Exemplary fusion proteins reported in the literature include fusions ofthe T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84(1987), 2936-2940; CD4 (Capon et al., Nature 337 (1989), 525-531;Traunecker et al., Nature 339 (1989), 68-70; Zettmeissl et al., DNA CellBiol. USA 9 (1990), 347-353; and Byrn et al., Nature 344 (1990),667-670); L-selectin (homing receptor) (Watson et al., J. Cell. Biol.110 (1990), 2221-2229; and Watson et al., Nature 349 (1991), 164-167);CD44 (Aruffo et al., Cell 61 (1990), 1303-1313); CD28 and B7 (Linsley etal., J. Exp. Med. 173 (1991), 721-730); CTLA-4 (Lisley et al., J. Exp.Med. 174 (1991), 561-569); CD22 (Stamenkovic et al., Cell 66 (1991),1133-1144); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88 (1991), 10535-10539; Lesslauer et al., Eur. J. Immunol. 27 (1991),2883-2886; and Peppel et al., J. Exp. Med. 174 (1991), 1483-1489 (1991);and IgE receptor a (Ridgway and Gorman, J. Cell. Biol. 115 (1991),Abstract No. 1448).

As discussed elsewhere herein, antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention may be fused toheterologous polypeptides to increase the in vivo half-life of thepolypeptides or for use in immunoassays using methods known in the art.For example, in one embodiment, PEG can be conjugated to the antibodiesof the invention to increase their half-life in vivo; see, e.g., Leonget al., Cytokine 16 (2001), 106-119; Adv. in Drug Deliv. Rev. 54 (2002),531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

Moreover, antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be fused to marker sequences,such as a peptide to facilitate their purification or detection. Inpreferred embodiments, the marker amino acid sequence is ahexa-histidine peptide (HIS), such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86 (1989), 821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37 (1984),767), GST, c-mycand the “flag” tag; see, e.g., Bill Brizzard,BioTechniques 44 (2008) 693-695 for a review of epitope taggingtechniques, and Table 1 on page 694 therein listing the most commonepitope tags usable in the present invention, the subject matter ofwhich is hereby expressly incorporated by reference.

Fusion proteins can be prepared using methods that are well known in theart; see for example U.S. Pat. Nos. 5,116,964 and 5,225,538. The precisesite at which the fusion is made may be selected empirically to optimizethe secretion or binding characteristics of the fusion protein. DNAencoding the fusion protein is then transfected into a host cell forexpression, which is performed as described hereinbefore.

Antibodies of the present invention may be used in non-conjugated formor may be conjugated to at least one of a variety of molecules, e.g., toimprove the therapeutic properties of the molecule, to facilitate targetdetection, or for imaging or therapy of the patient. Antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be labeled or conjugated either before or afterpurification, when purification is performed. In particular, antibodies,or antigen-binding fragments, variants, or derivatives thereof of theinvention may be conjugated to therapeutic agents, prodrugs, peptides,proteins, enzymes, viruses, lipids, biological response modifiers,pharmaceutical agents, or PEG.

Conjugates that are immunotoxins including conventional antibodies havebeen widely described in the art. The toxins may be coupled to theantibodies by conventional coupling techniques or immunotoxinscontaining protein toxin portions can be produced as fusion proteins.The antibodies of the present invention can be used in a correspondingway to obtain such immunotoxins. Illustrative of such immunotoxins arethose described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and byFanger, Immunol. Today 12 (1991), 51-54.

Those skilled in the art will appreciate that conjugates may also beassembled using a variety of techniques depending on the selected agentto be conjugated. For example, conjugates with biotin are prepared,e.g., by reacting an IAPP and/or proIAPP binding polypeptide with anactivated ester of biotin such as the biotin N-hydroxysuccinimide ester.Similarly, conjugates with a fluorescent marker may be prepared in thepresence of a coupling agent, e.g. those listed herein, or by reactionwith an isothiocyanate, preferably fluorescein-isothiocyanate.Conjugates of the antibodies, or antigen-binding fragments, variants orderivatives thereof of the invention are prepared in an analogousmanner.

The present invention further encompasses antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention conjugatedto a diagnostic or therapeutic agent. The antibodies can be useddiagnostically to, for example, demonstrate presence of a metabolicdisease, e.g., T2D to indicate the risk of getting a metabolic disease,to monitor the development or progression of a metabolic disease, i.e. adisease showing the occurrence of, or related to aggregated IAPP and/orproIAPP as part of a clinical testing procedure to, e.g., determine theefficacy of a given treatment and/or prevention regimen. In oneembodiment thus, the present invention relates to an antibody, which isdetectably labeled. Furthermore, in one embodiment, the presentinvention relates to an antibody, which is attached to a drug. Detectioncan be facilitated by coupling the antibody, or antigen-bindingfragment, variant, or derivative thereof to a detectable substance. Thedetectable substances or label may be in general an enzyme; a heavymetal, preferably gold; a dye, preferably a fluorescent or luminescentdye; or a radioactive label. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals using various positron emission tomographies, andnonradioactive paramagnetic metal ions; see, e.g., U.S. Pat. No.4,741,900 for metal ions which can be conjugated to antibodies for useas diagnostics according to the present invention. Examples of suitableenzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ¹¹¹Inor ⁹⁹Tc. Therefore, in one embodiment the present invention provides andetectably labeled antibody, wherein the detectable label is selectedfrom the group consisting of an enzyme, a radioisotope, a fluorophoreand a heavy metal

An antibody, or antigen-binding fragment, variant, or derivative thereofalso can be detectably labeled by coupling it to a chemiluminescentcompound. The presence of the chemiluminescent-tagged antibody is thendetermined by detecting the presence of luminescence that arises duringthe course of a chemical reaction. Examples of particularly usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.

One of the ways in which an antibody, or antigen-binding fragment,variant, or derivative thereof can be detectably labeled is by linkingthe same to an enzyme and using the linked product in an enzymeimmunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay(ELISA)” Microbiological Associates Quarterly Publication, Walkersville,Md., Diagnostic Horizons 2 (1978), 1-7); Voller et al., J. Clin. Pathol.31 (1978), 507-520; Butler, Meth. Enzymol. 73 (1981), 482-523; Maggio,E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980);Ishikawa, E. et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo(1981). The enzyme, which is bound to the antibody, will react with anappropriate substrate, preferably a chromogenic substrate, in such amanner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorimetric or by visual means. Enzymeswhich can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibody, orantigen-binding fragment, variant, or derivative thereof, it is possibleto detect the antibody through the use of a radioimmunoassay (RIA) (see,for example, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,(March, 1986)), which is incorporated by reference herein). Theradioactive isotope can be detected by means including, but not limitedto, a gamma counter, a scintillation counter, or autoradiography.

An antibody, or antigen-binding fragment, variant, or derivative thereofcan also be detectably labeled using fluorescence emitting metals suchas ¹⁵²Eu, or others of the lanthanide series. These metals can beattached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA). Techniques for conjugating various moieties to an antibody,or antigen-binding fragment, variant, or derivative thereof are wellknown, see, e.g., Anion et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), MarcelDekker, Inc., pp. 623-53 (1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62 (1982), 119-158.

As mentioned, in certain embodiments, a moiety that enhances thestability or efficacy of a binding molecule, e.g., a bindingpolypeptide, e.g., an antibody or immunospecific fragment thereof can beconjugated. For example, in one embodiment, PEG can be conjugated to thebinding molecules of the invention to increase their half-life in vivo.Leong et al., Cytokine 16 (2001), 106; Adv. in Drug Deliv. Rev. 54(2002), 531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

VI. Compositions and Methods of Use

The present invention relates to compositions comprising theaforementioned IAPP and/or proIAPP binding molecule, e.g., antibody orantigen-binding fragment thereof of the present invention or derivativeor variant thereof, or the polynucleotide, vector or cell of theinvention as defined hereinbefore. In one embodiment, the composition ofthe present invention is a pharmaceutical composition and furthercomprises a pharmaceutically acceptable carrier. Furthermore, thepharmaceutical composition of the present invention may comprise furtheragents such as interleukins or interferons depending on the intended useof the pharmaceutical composition. For use in the treatment of ametabolic disease showing the occurrence of, or related to aggregatedIAPP and/or proIAPP, e.g., of T2D, the additional agent may be selectedfrom the group consisting of small organic molecules, anti-IAPP and/oranti-proIAPP antibodies, and combinations thereof. Hence, in aparticular preferred embodiment the present invention relates to the useof the IAPP and/or proIAPP binding molecule, e.g., antibody orantigen-binding fragment thereof of the present invention or of abinding molecule having substantially the same binding specificities ofany one thereof, the polynucleotide, the vector or the cell of thepresent invention for the preparation of a pharmaceutical or diagnosticcomposition for prophylactic and therapeutic treatment of a metabolicdisease, monitoring the progression of a metabolic disease or a responseto a metabolic disease treatment in a subject or for determining asubject's risk for developing a metabolic disease.

Hence, in one embodiment the present invention relates to a method oftreating a metabolic disorder characterized by abnormal accumulationand/or deposition of IAPP and/or proIAPP in islets of Langerhans, whichmethod comprises administering to a subject in need thereof atherapeutically effective amount of any one of the afore-described IAPPand/or proIAPP binding molecules, antibodies, polynucleotides, vectorsor cells of the instant invention. The terms “metabolic disorder”includes but is not limited to the group of disorders generallycharacterized by symptoms such as metabolic changes preceding, causing,and/or connected/associated with or linked to T2D comprising diseasesthat cause damage to the pancreas and could therefore lead to diabetescomprising chronic pancreatitis, cystic fibrosis, pancreatic cancer; indiseases that increase the risk of T2D comprising Alzheimer's disease,Huntington's disease; in cardiovascular diseases linked or not withobesity and T2D; and/or to T2D itself.

A particular advantage of the therapeutic approach of the presentinvention lies in the fact that the antibodies of the present inventionare derived from B cells or B memory cells from healthy human subjectswith no signs of a disease showing the occurrence of, or related toaggregated IAPP and/or proIAPP such as T2D and thus are, with a certainprobability, capable of preventing a clinically manifest disease relatedto aggregated IAPP and/or proIAPP, or of diminishing the risk of theoccurrence of the clinically manifest disease, or of delaying the onsetor progression of the clinically manifest disease. Typically, theantibodies of the present invention also have already successfully gonethrough somatic maturation, i.e. the optimization with respect toselectivity and effectiveness in the high affinity binding to the targetIAPP and/or proIAPP molecule by means of somatic variation of thevariable regions of the antibody.

The knowledge that such cells in vivo, e.g. in a human, have not beenactivated by means of related or other physiological proteins or cellstructures in the sense of an autoimmunological or allergic reaction isalso of great medical importance since this signifies a considerablyincreased chance of successfully living through the clinical testphases. So to speak, efficiency, acceptability and tolerability havealready been demonstrated before the preclinical and clinicaldevelopment of the prophylactic or therapeutic antibody in at least onehuman subject. It can thus be expected that the human anti-IAPP and/oranti-proIAPP antibodies of the present invention, both its targetstructure-specific efficiency as therapeutic agent and its decreasedprobability of side effects significantly increase its clinicalprobability of success.

The present invention also provides a pharmaceutical and diagnostic,respectively, pack or kit comprising one or more containers filled withone or more of the above described ingredients, e.g. anti-IAPP and/oranti-proIAPP antibody, binding fragment, derivative or variant thereof,polynucleotide, vector or cell of the present invention. Associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition or alternatively the kit comprises reagents and/or instructionsfor use in appropriate diagnostic assays. The composition, e.g. kit ofthe present invention is of course particularly suitable for the riskassessment, diagnosis, prevention and treatment of a disorder which isaccompanied with the presence of aggregated IAPP and/or proIAPP, and inparticular applicable for the treatment of disorders generallycharacterized by symptoms such as metabolic changes preceding, causing,and/or connected/associated with or linked to T2D comprising diseasesthat cause damage to the pancreas and could therefore lead to diabetescomprising chronic pancreatitis, cystic fibrosis, pancreatic cancer; indiseases that increase the risk of T2D comprising Alzheimer's disease,Huntington's disease; in cardiovascular diseases linked or not withobesity and T2D; and/or to T2D itself, for example.

The pharmaceutical compositions of the present invention can beformulated according to methods well known in the art; see for exampleRemington: The Science and Practice of Pharmacy (2000) by the Universityof Sciences in Philadelphia, ISBN 0-683-306472. Examples of suitablepharmaceutical carriers are well known in the art and include phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc.Compositions comprising such carriers can be formulated by well-knownconventional methods. These pharmaceutical compositions can beadministered to the subject at a suitable dose. Administration of thesuitable compositions may be effected by different ways, e.g., byintravenous, intraperitoneal, subcutaneous, intramuscular, intranasal,topical or intradermal administration or spinal or brain delivery.Aerosol formulations such as nasal spray formulations include purifiedaqueous or other solutions of the active agent with preservative agentsand isotonic agents. Such formulations are preferably adjusted to a pHand isotonic state compatible with the nasal mucous membranes.Formulations for rectal or vaginal administration may be presented as asuppository with a suitable carrier.

The dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. A typical dose can be, for example, in therange of 0.001 to 1000 μg (or of nucleic acid for expression or forinhibition of expression in this range); however, doses below or abovethis exemplary range are envisioned, especially considering theaforementioned factors. Generally, the dosage can range, e.g., fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), ofthe host body weight. For example dosages can be 1 mg/kg body weight or10 mg/kg body weight or within the range of 1-10 mg/kg, preferably atleast 1 mg/kg. Doses intermediate in the above ranges are also intendedto be within the scope of the invention. Subjects can be administeredsuch doses daily, on alternative days, weekly or according to any otherschedule determined by empirical analysis. An exemplary treatmententails administration in multiple dosages over a prolonged period, forexample, of at least six months. Additional exemplary treatment regimensentail administration once per every two weeks or once a month or onceevery 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kgweekly. In some methods, two or more monoclonal antibodies withdifferent binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated. Progress can be monitored by periodic assessment.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringers dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Furthermore, the pharmaceutical composition of theinvention may comprise further agents such as dopamine orpsychopharmacologic drugs, depending on the intended use of thepharmaceutical composition.

Furthermore, in a preferred embodiment of the present invention thepharmaceutical composition may be formulated as a vaccine, for example,if the pharmaceutical composition of the invention comprises ananti-IAPP and/or anti-proIAPP antibody or binding fragment, derivativeor variant thereof for passive immunization. As mentioned in thebackground section, several lines of evidence have been shown indicatingthat aggregated IAPP and/or proIAPP species are a major trigger for T2Dpathogenesis (Zraika et al. (2010), Diabetologia 53(6): 1046-1056;Westermark et al. (2011), Physiol. Rev. 91(3): 795-826; Jurgens et al.(2011), Am. J. Pathol. 178(6): 2632-2640; Hoppener et al. (2008), Exp.Diabetes Res. 697035) and treatment interfering with hIAPP aggregationameliorated the diabetic phenotype and increased animal life span(Aitken et al. (2009), Diabetes 59(1): 161-171). Accordingly, it isprudent to expect that passive immunization with human anti-IAPP and/oranti-proIAPP antibodies and equivalent IAPP and/or proIAPP bindingmolecules of the present invention will help to circumvent severaladverse effects of active immunization therapy concepts as alreadydiscussed in the background section. Therefore, the present anti-IAPPand/or anti-proIAPP antibodies and their equivalents of the presentinvention will be particularly useful as a vaccine for the prevention oramelioration of diseases showing the presence of, or caused byaggregated IAPP and/or pro/IAPP such as metabolic changes preceding,causing, and/or connected/associated with or linked to T2D comprisingdiseases that cause damage to the pancreas and could therefore lead todiabetes comprising chronic pancreatitis, cystic fibrosis, pancreaticcancer; in diseases that increase the risk of T2D comprising Alzheimer's disease, Huntington's disease; in cardiovascular diseases linked ornot with obesity and T2D; and/or to T2D itself, for example.

In one embodiment, it may be beneficial to use recombinant bispecific ormultispecific constructs of the antibody of the present invention. For areference see Fischer and Leger, Pathobiology 74 (2007), 3-14. Suchbispecific molecule might be designed to target IAPP with one bindingarm and another entity known in pathogenesis of diabetes with the secondarm, e.g., proIAPP (besides antibodies of the present invention whichare bispecific against IAPP and proIAPP as indicated above for theexemplary antibody NI-203.26C11). Or such a bispecific molecule might bedesigned to bind with the second binding arm other entities known inpathogenesis of diabetes such as IL-1β and IL-6, or by blockingsodium-glucose cotransporter-2 (SGLT2) or CD33, which intervention isthought to reverse diabetes in NOD mice by induction of adaptiveregulatory T cells (Ablamunits et al., Diabetes 61 (2012), 145-154;Belghith et al., Nat Med 9 (2003), 1202-1208).

In one embodiment, it may be beneficial to use recombinant Fab (rFab)and single chain fragments (scFvs) of the antibody of the presentinvention, which might more readily penetrate a cell membrane. Forexample, Robert et al., Protein Eng. Des. Sel. (2008) Oct. 16;S1741-0134, published online ahead, describe the use of chimericrecombinant Fab (rFab) and single chain fragments (scFvs) of monoclonalantibody WO-2 which recognizes an epitope in the N-terminal region ofAβ. The engineered fragments were able to (i) prevent amyloidfibrillization, (ii) disaggregate preformed A131-42 fibrils and (iii)inhibit A131-42 oligomer-mediated neurotoxicity in vitro as efficientlyas the whole IgG molecule. The perceived advantages of using small Faband scFv engineered antibody formats which lack the effector functioninclude more efficient passage across the blood-brain barrier andminimizing the risk of triggering inflammatory side reactions.Furthermore, besides scFv and single-domain antibodies retain thebinding specificity of full-length antibodies, they can be expressed assingle genes and intracellularly in mammalian cells as intrabodies, withthe potential for alteration of the folding, interactions,modifications, or subcellular localization of their targets; see forreview, e.g., Miller and Messer, Molecular Therapy 12 (2005), 394-401.

In a different approach Muller et al., Expert Opin. Biol. Ther. (2005),237-241, describe a technology platform, so-called ‘SuperAntibodyTechnology’, which is said to enable antibodies to be shuttled intoliving cells without harming them. Such cell-penetrating antibodies opennew diagnostic and therapeutic windows. The term ‘TransMabs’ has beencoined for these antibodies.

In a further embodiment, co-administration or sequential administrationof other antibodies useful for treating a disease related to theoccurrence of aggregated IAPP and/or proIAPP may be desirable. In oneembodiment, the additional antibody is comprised in the pharmaceuticalcomposition of the present invention. Examples of antibodies which canbe used to treat a subject include, but are not limited to, antibodiestargeting CD33, SGLT2, IL-6 and IL-1.

In a further embodiment, co-administration or sequential administrationof other agents useful for treating a disease related to aggregated IAPPand/or proIAPP, and/or to diabetes may be desirable. In one embodiment,the additional agent is comprised in the pharmaceutical composition ofthe present invention. Examples of agents which can be used to treat asubject include, but are not limited to: Insulin and insulin analogues;insulin signaling pathway modulators, such as inhibitors of proteintyrosine phosphatases (PTPases), non-small molecule mimetic compoundsand inhibitors of glutamine-fructose-6-phosphate amidotransferase(GFAT), DPP-IV inhibitors, agents influencing a deregulated hepaticglucose production, like inhibitors of glucose-6-phosphatase (G6Pase),inhibitors of fructose-1,6-bisphosphatase (F-1,6-BPase), inhibitors ofglycogen phosphorylase (GP), glucagon receptor antagonists, inhibitorsof phosphoenolpyruvate carboxykinase (PEPCK), pyruvate dehydrogenasekinase (PDHK) inhibitors, insulin sensitivity enhancers, insulinsecretion enhancers, α-glucosidase inhibitors, inhibitors of gastricemptying; Glucagon-Like-Peptide-1 (GLP-1) receptor agonists;Sulfonylurea agents; Biguanide agents such as Metformin;Alpha-glucosidase inhibitors; peroxisome proliferator-activated receptor(PPAR)-Agonists; Meglitinide agents; Dipeptidyl-peptidase (DPP) IVinhibitors; PDE1, PDE5, PDE9, PDE10 or PDE11 (PDE=Phosphodiesterase)inhibitors; Amylin agonists (e.g., pramlintide and other amylinanalogues); Cinnamon; Glucagon receptor antagonists;Glycogen-Phosphorylase inhibitors; Fructose-1,6-Bisphosphate inhibitors;Cannabinoid (CB1) receptor antagonists; Anti-obesity drugs such asappetite suppressors, satiety increasing substances, and energyexpenditure increasing drugs; anti-inflammatory agents or anycombination thereof. Examples of agents which may be used for treatingor preventing islet rejection following clinical pancreatic islettransplantation include but are not limited to the agents of the groupcomprising sirolimus (rapamycin), Calcineurin inhibitors (e.g.,tacrolimus), cyclosporine, mycophenolate mofetil, FTY 720, cyclosporine,corticosteroides and anti-IL2-receptor monoclonal antibodies (e.g.,daclizumab), glucagon-like peptide-1 (GLP-1) receptor agonists (see,e.g., Noguchi et al., Acta Med. Okayama, 60 (2006), and theinternational application WO2012088157). Therefore, in one embodiment acomposition is provided further comprising an additional agent usefulfor treating diabetes mellitus type 2 (T2D) and/or in treating orpreventing islet rejection following clinical pancreatic islettransplantation. Examples of other agents that may be used concomitantwith a pharmaceutical composition of the present invention are describedin the art; see, e.g. international applications WO2009005672,WO2010128092, WO2012088157 or European application EP11158212.8.

A therapeutically effective dose or amount refers to that amount of theactive ingredient sufficient to ameliorate the symptoms or condition.Therapeutic efficacy and toxicity of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Preferably, thetherapeutic agent in the composition is present in an amount sufficientto restore or preserve normal blood sugar control and/or insulinresponse in case of metabolic disorders such as T2D.

From the foregoing, it is evident that the present invention encompassesany use of an IAPP and/or proIAPP binding molecule comprising at leastone CDR of the above described antibody, in particular for diagnosingand/or treatment of a disease related to aggregated IAPP and/or proIAPPas mentioned above, such as T2D. Preferably, said binding molecule is anantibody of the present invention or an immunoglobulin chain thereof. Inaddition, the present invention relates to anti-idiotypic antibodies ofany one of the mentioned antibodies described hereinbefore. These areantibodies or other binding molecules which bind to the unique antigenicpeptide sequence located on an antibody's variable region near theantigen-binding site and are useful, e.g., for the detection ofanti-IAPP and/or anti-proIAPP antibodies in a sample obtained from asubject. In one embodiment thus, the present invention provides anantibody as defined hereinabove and below or an IAPP and/or proIAPPbinding molecule having substantially the same binding specificities ofany one thereof, the polynucleotide, the vector or the cell as definedherein or a pharmaceutical or diagnostic composition comprising any onethereof for use in prophylactic treatment, therapeutic treatment and/ormonitoring the progression or a response to treatment of a disorderrelated to IAPP and/or proIAPP, preferably wherein the disorder isselected from the group comprising all types of diabetes, such as type 1diabetes, gestational diabetes, prediabetes (when high blood glycemia isnot reaching the T2D threshold or insulin resistance) and latentautoimmune diabetes of adults (LADA); any disease that causes damage tothe pancreas and could therefore lead to diabetes such as chronicpancreatitis, cystic fibrosis and pancreatic cancer; any disease thatincreases the risk of T2D such as Alzheimer's disease and Huntington'sdisease or other neurodegenerative diseases which have been definedherein as associated with diabetes; metabolic syndrome in general as arisk factor for developing diabetes or as a condition that could existprior diabetes; islet amyloidosis in general as a risk factor fordeveloping diabetes or as a condition that could exist prior diabetes;obesity in general as a risk factor for developing diabetes or as acondition that could exist prior diabetes; any cardiovascular diseaselinked or not with obesity and T2D; all the consequences of T2D that mayalso increase the risk of developing diabetes such heart disease,strokes, diabetic retinopathy, kidney failure, renal failure,ketoacidosis and nonketotic hyperosmolar coma. The above group ofdisorders will be referred to as the group of disorders related to IAPPand/or proIAPP.

In another embodiment the present invention relates to a diagnosticcomposition comprising any one of the above described IAPP and/orproIAPP binding molecules, antibodies, antigen-binding fragments,polynucleotides, vectors or cells of the invention and optionallysuitable means for detection such as reagents conventionally used inimmuno- or nucleic acid-based diagnostic methods. The antibodies of theinvention are, for example, suited for use in immunoassays in which theycan be utilized in liquid phase or bound to a solid phase carrier.Examples of immunoassays which can utilize the antibody of the inventionare competitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA), the sandwich (immunometric assay), flow cytometry and the Westernblot assay. The antigens and antibodies of the invention can be bound tomany different carriers and used to isolate cells specifically boundthereto. Examples of well-known carriers include glass, polystyrene,polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran,nylon, amyloses, natural and modified celluloses, polyacrylamides,agaroses, and magnetite. The nature of the carrier can be either solubleor insoluble for the purposes of the invention. There are many differentlabels and methods of labeling known to those of ordinary skill in theart. Examples of the types of labels which can be used in the presentinvention include enzymes, radioisotopes, colloidal metals, fluorescentcompounds, chemiluminescent compounds, and bioluminescent compounds; seealso the embodiments discussed hereinabove.

By a further embodiment, the IAPP and/or proIAPP binding molecules, inparticular antibodies of the present invention may also be used in amethod for the diagnosis of a disorder in an individual by obtaining abody fluid sample from the tested individual which may be a bloodsample, a plasma sample, a serum sample, a lymph sample or any otherbody fluid sample, such as a saliva or a urine sample and contacting thebody fluid sample with an antibody of the instant invention underconditions enabling the formation of antibody-antigen complexes. Thelevel of such complexes is then determined by methods known in the art,a level significantly higher than that formed in a control sampleindicating the disease in the tested individual. In the same manner, thespecific antigen bound by the antibodies of the invention may also beused. Thus, the present invention relates to an in vitro immunoassaycomprising the binding molecule, e.g., antibody or antigen-bindingfragment thereof of the invention.

In this context, the present invention also relates to meansspecifically designed for this purpose. For example, an antibody-basedarray may be used, which is for example loaded with antibodies orequivalent antigen-binding molecules of the present invention whichspecifically recognize IAPP and/or proIAPP. Design of microarrayimmunoassays is summarized in Kusnezow et al., Mol. Cell Proteomics 5(2006), 1681-1696. Accordingly, the present invention also relates tomicroarrays loaded with IAPP and/or proIAPP binding molecules identifiedin accordance with the present invention.

In one embodiment, the present invention relates to a method ofdiagnosing a disease related to aggregated IAPP and/or proIAPP in asubject, the method comprising determining the presence of IAPP and/orproIAPP and/or aggregated IAPP and/or proIAPP in a sample from thesubject to be diagnosed with at least one antibody of the presentinvention, an IAPP and/or proIAPP binding fragment thereof or an IAPPand/or proIAPP-binding molecule having substantially the same bindingspecificities of any one thereof, wherein the presence of pathologicallyaggregated IAPP and/or proIAPP is indicative of a metabolic disorder,such as T2D and an increase of the level of the pathologicallyaggregated IAPP and/or proIAPP in comparison to the level of thephysiological IAPP and/or proIAPP monomeric forms is indicative forprogression of a metabolic disorder in said subject.

The subject to be diagnosed may be asymptomatic or preclinical for thedisease. Preferably, the control subject has a disease related toaggregated IAPP and/or proIAPP, for example, metabolic changespreceding, causing, and/or connected/associated with or linked to T2Dcomprising diseases that cause damage to the pancreas and couldtherefore lead to diabetes comprising chronic pancreatitis, cysticfibrosis, pancreatic cancer; in diseases that increase the risk of T2Dcomprising Alzheimer's disease, Huntington's disease; in cardiovasculardiseases linked or not with obesity and T2D; and/or to T2D itself,wherein a similarity between the level of pathologically aggregated IAPPand/or proIAPP and the reference standard indicates that the subject tobe diagnosed has a metabolic or is at risk to develop a metabolicdisease. Alternatively, or in addition as a second control the controlsubject does not have a metabolic disease, wherein a difference betweenthe level of physiological IAPP and/or proIAPP monomers and/or ofaggregated IAPP and/or proIAPP and the reference standard indicates thatthe subject to be diagnosed has a metabolic disease or is at risk todevelop a metabolic disease. Preferably, the subject to be diagnosed andthe control subject(s) are age-matched. The sample to be analyzed may beany body fluid suspected to contain pathologically aggregated IAPPand/or proIAPP, for example a blood, blood plasma, blood serum, urine,peritoneal fluid, saliva or cerebral spinal fluid (CSF).

The level of physiological IAPP and/or proIAPP monomers and/or ofpathologically aggregated IAPP and/or proIAPP may be assessed by anysuitable method known in the art comprising, e.g., analyzing IAPP and/orproIAPP by one or more techniques chosen from Western blot,immunoprecipitation, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), fluorescent activated cell sorting (FACS),two-dimensional gel electrophoresis, mass spectroscopy (MS),matrix-assisted laser desorption/ionization-time of flight-MS(MALDI-TOF), surface-enhanced laser desorption ionization-time of flight(SELDI-TOF), high performance liquid chromatography (HPLC), fast proteinliquid chromatography (FPLC), multidimensional liquid chromatography(LC) followed by tandem mass spectrometry (MS/MS), and laserdensitometry. Preferably, said in vivo imaging of IAPP and/or proIAPPcomprises positron emission tomography (PET), single photon emissiontomography (SPECT), near infrared (NIR) optical imaging or magneticresonance imaging (MRI).

Antibody based methods for detection of IAPP and/or proIAPP and fordiagnosing or monitoring the progression of a disease related toaggregated IAPP and/or proIAPP such as T2D, and monitoring the treatmentof such a disease using antibodies and related means which may beadapted in accordance with the present invention are also described ininternational application WO2003092619 the disclosure content of whichbeing incorporated herein by reference. Those methods may be applied asdescribed but with an IAPP and/or proIAPP specific antibody, bindingfragment, derivative or variant of the present invention.

In one embodiment thus, an antibody of the present invention or an IAPPand/or proIAPP binding molecule having substantially the same bindingspecificities of any one thereof, the polynucleotide, the vector or thecell as defined hereinabove or a pharmaceutical or diagnosticcomposition comprising any one thereof is provided for use inprophylactic treatment, therapeutic treatment and/or monitoring theprogression or a response to treatment of a disorder related to IAPPand/or proIAPP. In general thus, the present invention also relates to amethod of diagnosing or monitoring the progression of a disorder relatedto IAPP and/or proIAPP (such as islet amyloidosis and T2D which isusually preceded by islet amyloidosis) in a subject, the methodcomprising determining the presence of IAPP and/or proIAPP oligomers,aggregates or fibrils in a sample from the subject to be diagnosed withat least one antibody of the present invention or an IAPP and/or proIAPPbinding molecule having substantially the same binding specificities ofany one thereof, wherein the presence of IAPP and/or proIAPP oligomers,aggregates or fibrils is indicative of the disorder. In one embodimentsaid method of diagnosing or monitoring the progression of isletamyloidosis in a subject is provided, the method comprising determiningthe presence of IAPP and/or proIAPP oligomers, aggregates or fibrils ina sample from the subject to be diagnosed with at least one antibody ofthe present invention or an IAPP and/or proIAPP binding molecule havingsubstantially the same binding specificities of any one thereof, whereinthe presence of IAPP and/or proIAPP oligomers, aggregates or fibrils isindicative of presymptomatic, prodromal or clinical diabetes mellitustype 2 (T2D) and/or of beta-cell failure following clinical pancreaticislet transplantation and an increase of the level of IAPP and/orproIAPP oligomers, aggregates or fibrils in comparison to the level ofthe physiological IAPP or in comparison to a reference sample derivedfrom a healthy control subject or a control sample from the same subjectis indicative for progression of presymptomatic, prodromal orestablished diabetes mellitus type 2 (T2D) and/or of islet failurefollowing clinical pancreatic islet transplantation in said subject. Itwould be appreciated by any person skilled in the art that in oneembodiment said method is used as well for the diagnosing or monitoringthe progression of any other disorder from the group of disordersrelated to IAPP and/or proIAPP as defined hereinabove.

As indicated above, the antibodies of the present invention, fragmentsthereof and molecules of the same binding specificity as the antibodiesand fragments thereof may be used not only in vitro but in vivo as well,wherein besides diagnostic, therapeutic applications as well may bepursued. In one embodiment thus, the present invention also relates toan IAPP and/or proIAPP binding molecule comprising at least one CDR ofan antibody of the present invention for the preparation of acomposition for in vivo detection of or targeting a therapeutic and/ordiagnostic agent to IAPP and/or proIAPP in the human or animal body.Potential therapeutic and/or diagnostic agents may be chosen from thenonexhaustive enumerations of the therapeutic agents useful in treatmentof metabolic diseases, such as T2D and potential labels as indicatedhereinbefore. In respect of the in vivo imaging, in one preferredembodiment the present invention provides said IAPP and/or proIAPPbinding molecule comprising at least one CDR of an antibody of thepresent invention, wherein said in vivo imaging comprises positronemission tomography (PET), single photon emission tomography (SPECT),near infrared (NIR) optical imaging or magnetic resonance imaging (MRI).In a further embodiment the present invention also provides said IAPPand/or proIAPP binding molecule comprising at least one CDR of anantibody of the present invention, or said molecule for the preparationof a composition for the above specified in vivo imaging methods, forthe use in the method of diagnosing or monitoring the progression of adisorder related to IAPP and/or proIAPP in a subject, as definedhereinabove.

VII. Peptides with Aggregation Specific IAPP Epitopes

In a further aspect the present invention relates to peptides having anepitope of IAPP and/or proIAPP specifically recognized by any antibodyof the present invention. Preferably, such peptide comprises or consistsof an amino acid sequence as indicated in SEQ ID NO: 4, in SEQ ID NO: 5,or in SEQ ID NO: 71 as the unique linear epitope recognized by theantibody or a modified sequence thereof in which one or more amino acidsare substituted, deleted and/or added, wherein the peptide is recognizedby any antibody of the present invention, preferably by antibodyNI-203.19H8 respective by antibody NI-203.26C11 or by antibodyNI-203.15C7.

In one embodiment of this invention such a peptide may be used fordiagnosing or monitoring a disease related to aggregated IAPP and/orproIAPP in a subject, such as T2D comprising a step of determining thepresence of an antibody that binds to a peptide in a biological sampleof said subject, and being used for diagnosis of such a disease in saidsubject by measuring the levels of antibodies which recognize the abovedescribed peptide of the present invention and comparing themeasurements to the levels which are found in healthy subjects ofcomparable age and gender. Thus in one embodiment the present inventionrelates to a method for diagnosing islet amyloidosis indicative ofpresymptomatic or clinical diabetes mellitus type 2 (T2D) and/or ofbeta-cell failure following clinical pancreatic islet transplantation ina subject, comprising a step of determining the presence of an antibodythat binds to a peptide as defined above in a biological sample of saidsubject. According to this method, an elevated level of measuredantibodies specific for said peptide of the present invention isindicative for diagnosing in said subject presymptomatic or clinicaldiabetes mellitus type 2 (T2D) and/or of beta-cell failure followingclinical pancreatic islet transplantation or for diagnosing in saidsubject any other disease from the group of disorders related to IAPPand/or proIAPP as defined hereinabove. The peptide of the presentinvention may be formulated in an array, a kit and composition,respectively, as described hereinbefore. In this context, the presentinvention also relates to a kit useful in the diagnosis or monitoringthe progression of islet amyloidosis, said kit comprising at least oneantibody of the present invention or an IAPP and/or proIAPP bindingmolecule having substantially the same binding specificities of any onethereof, the polynucleotide, the vector or the cell and/or the peptideas respectively defined hereinbefore, optionally with reagents and/orinstructions for use.

These and other embodiments are disclosed and encompassed by thedescription and examples of the present invention. Further literatureconcerning any one of the materials, methods, uses and compounds to beemployed in accordance with the present invention may be retrieved frompublic libraries and databases, using for example electronic devices.For example the public database “Medline” may be utilized, which ishosted by the National Center for Biotechnology Information and/or theNational Library of Medicine at the National Institutes of Health.Further databases and web addresses, such as those of the EuropeanBioinformatics Institute (EBI), which is part of the European MolecularBiology Laboratory (EMBL) are known to the person skilled in the art andcan also be obtained using internet search engines. An overview ofpatent information in biotechnology and a survey of relevant sources ofpatent information useful for retrospective searching and for currentawareness is given in Berks, TIBTECH 12 (1994), 352-364.

The above disclosure generally describes the present invention. Unlessotherwise stated, a term as used herein is given the definition asprovided in the Oxford Dictionary of Biochemistry and Molecular Biology,Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 019 850673 2. Several documents are cited throughout the text of thisspecification. Full bibliographic citations may be found at the end ofthe specification immediately preceding the claims. The contents of allcited references (including literature references, issued patents,published patent applications as cited throughout this application andmanufacturer's specifications, instructions, etc.) are hereby expresslyincorporated by reference; however, there is no admission that anydocument cited is indeed prior art as to the present invention.

A more complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only and are not intended to limit the scope of theinvention.

EXAMPLES Example 1: Validation of Target and Binding Specificity ofHuman IAPP Antibodies

To validate IAPP as a recognized target of isolated antibodies, directELISA assays were performed. For the exemplary recombinant humanNI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3 antibodies, 96-wellmicroplates (Costar, Corning, USA) were coated with human IAPP solutionor with BSA (Sigma-Aldrich, Buchs, Switzerland) diluted to aconcentration of 10 μg/ml in carbonate ELISA coating buffer (15 mMNa₂CO₃, 35 mM NaHCO₃, pH 9.42) and binding efficiency of the antibodywas tested. Importantly, the human IAPP solution used for ELISA assaycontained IAPP fibrils, as shown by electron microscopy; see FIG. 3A.The exemplary NI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3antibodies specifically bind to human IAPP fibrils by ELISA. No bindingis observed to BSA; see FIG. 3B. The same characteristics seem to applyto the antibodies NI-203.19F2 and NI-203.15C7.

For a determination of the half maximal effective concentration (EC₅₀)of the exemplary antibodies NI-203.9A2, NI-203.19H8, NI-203.26C11 andNI-203.8E3, additional direct ELISA experiments with varying antibodyconcentrations were performed. 96-well microplates (Costar, Corning,USA) were coated with human IAPP and human proIAPP solutions diluted toa concentration of 10 μg/ml in carbonate ELISA coating buffer (15 mMNa₂CO₃, 35 mM NaHCO₃, pH 9.42) and binding efficiency of the antibodywas tested. While the human IAPP solution used for ELISA assay containsIAPP fibrils, human proIAPP formed large aggregates in solution, asrevealed by electron microscopy; see FIG. 4A. Binding was determinedusing a donkey anti-human IgGγ antibody (Jackson ImmunoResearch,Newmarket, UK) conjugated with HRP, followed by measurement of HRPactivity in a standard colorimetric assay.

The EC₅₀ values were estimated by a non-linear regression using GraphPadPrism (San Diego, USA) software. Recombinant human-derived antibodiesNI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3 bind with a highaffinity to human IAPP fibrils with an EC₅₀ of 9 nM, 22 nM, 6 nM and 4nM, respectively. Antibody NI-203.26C11 also binds to aggregated humanproIAPP with an EC₅₀ in the nanomolar range (260 nM); see FIG. 4B.

Example 2: Antibody Specificity to Human IAPP Fibrils and not toNonfibrillar Human IAPP Thus Preferably Binding to ConformationalEpitopes

To determine the binding capacity of the exemplary NI-203.9A2,NI-203.19H8, NI-203.26C11 and NI-203.8E3 antibodies to conformationalepitopes, direct ELISA experiments were performed with human IAPP andnonfibrillar IAPP solutions diluted to a concentration of 10 μg/ml incarbonate ELISA coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.42) andbinding capacity of the antibody was tested. While the human IAPPsolution used for ELISA assay contains IAPP fibrils, human nonfibrillarIAPP solution was lacking IAPP fibrils and only showed small amorphousaggregates, as revealed by electron microscopy; see FIG. 5A. Binding wasdetermined using a donkey anti-human IgGγ antibody (JacksonimmunoResearch, Newmarket, UK) conjugated with HRP, followed bymeasurement of HRP activity in a standard colorimetric assay.

Recombinant NI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3antibodies showed high affinity binding to IAPP fibrils upon coatingwith the IAPP solution (FIG. 5B), as previously observed (FIG. 4). Aloss in affinity was observed on nonfibrillar IAPP when compared to IAPPfibrils (FIG. 5B), thus demonstrating preferential binding ofNI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3 antibodies to IAPPfibrils. Preliminary results show the same effects as described abovefor antibodies NI-203.19F2 and NI-203.15C7.

These findings strongly point to NI-203.9A2, NI-203.19H8, NI-203.26C11,NI-203.8E3, NI-203.19F2 and NI-203.15C7 antibody binding epitopes thatare predominantly exposed and accessible upon IAPP fibril formation, incontrast to linear epitopes that are present in the physiological humanIAPP protein conformation. Pathological IAPP fibrils are observed inpancreatic islets of T2D patients. Since NI-203.9A2, NI-203.19H8,NI-203.26C11, NI-203.8E3, NI-203.19F2 and NI-203.15C7 antibodies showprominent binding to therapeutically relevant pathological human IAPPfibrils, these human-derived antibodies are of therapeutic potential inT2D.

Example 3: Assessment of the Binding Epitope of NI-203.9A2, NI-203.19H8,NI-203.26C11, NI-203.8E3, NI-203.19F2, and NI-203.15C7 Antibodies

To determine the binding epitope of the exemplary NI-203.9A2,NI-203.19H8, NI-203.26C11, NI-203.8E3, NI-203.19F2, and NI-203.15C7antibodies, pepscan and alanine scan analysis was performed withoverlapping peptides mapping the entire human IAPP amino acid sequenceand with alanine substitution on the first 22 amino acids of proIAPP.Binding capacity of the antibody was tested on these peptides spottedonto a nitrocellulose membrane (JPT Peptide Technologies, Berlin,Germany) and using HRP-conjugated donkey anti-human IgGγ secondaryantibody (Jackson immunoResearch, Newmarket, UK) followed by detectionof HRP activity (FIG. 6A).

Recombinant NI-203.19H8, NI-203.26C11, and NI-203.15C7 antibodies (1μg/ml) showed binding to the sequence 19-SSNNFGA-25 (SEQ ID NO: 4),2-CNTATCA-8 (SEQ ID NO: 5), and 10-QRLANFLVHS-19 (SEQ ID NO: 71) onhuman IAPP (FIGS. 6A and 6B), thus corresponding to the putative bindingepitope sequences of these antibodies. The epitope of recombinantNI-203.9A2, NI-203.8E3, and NI-203.19F2 antibodies (1 and 10 μg/ml) havenot been identified.

Example 4: Binding of NI-203.9A2, NI-203.19H8 and NI-203.26C11Antibodies to Pathological IAPP Fibrils in the Pancreas of PatientsDiagnosed with Diabetes Mellitus Type 2 but not in Control Patients

Paraffin-embedded pancreas sections of two patients diagnosed withdiabetes mellitus type 2 (T2D) were selected based on amyloid load inpancreatic islets observed upon ThioS and Congo red staining, andsubsequently used for the exemplary NI-203.9A2, NI-203.19H8 andNI-203.26C11 antibody binding characterization. Paraffin-embeddedpancreas sections of a patient not diagnosed with diabetes mellitus type2 were used as control. After formic acid pretreatment, sections wereincubated with human NI-203.9A2, NI-203.19H8 and NI-203.26C11 antibodies(5 and 50 nM) or mouse monoclonal anti-IAPP antibody (1:100; Abcam,Cambridge, UK), followed by incubation with biotinylated donkeyanti-human secondary antibody (1:500; Jackson ImmunoResearch, Newmarket,UK) or biotinylated goat anti-mouse secondary antibody (1:500; JacksonImmunoResearch, Newmarket, UK). Antibody signal was amplified with theVectastain ABC-AP kit (Vector Laboratories, USA) and detected withdiaminobenzidine substrate (Thermo Fisher Scientific, USA). Uponavidin/biotin blocking (Avidin/Biotin blocking kit, Vector Laboratories,USA), pancreatic islet β-cells were visualized using a polyclonal guineapig anti-insulin antibody (1:5; Dako, USA) coupled to a biotinylateddonkey anti-guinea pig secondary antibody (1:500; Jackson ImmunoResearchLaboratories, USA) and antibody signal was amplified with the VectastainABC-AP kit (Vector Laboratories, USA) and detected with alkalinephosphatase substrate (Vector Laboratories, USA).

The first T2D patient showed large amyloid deposits in pancreatic isletscorresponding to pathological IAPP fibrils, as visualized by ThioS andCongo red staining (FIG. 7A). NI-203.9A2, NI-203.19H8 and NI-203.26C11human antibodies showed prominent pancreatic islet staining on theseamyloid-positive sections (FIG. 7B). The antibody staining was observedat 5 nM and increased at 50 nM, with no staining observed with thesecondary antibody only, suggesting specific binding of the human IAPPantibodies. These findings were confirmed on a second T2D patientshowing amyloid deposits in pancreatic islets (data not shown). Incontrast, NI-203.9A2, NI-203.19H8 and NI-203.26C11 human antibodies werenot showing any staining on pancreatic islets from a third T2D patientlacking amyloid deposits (FIGS. 7C and D) and from a control patient notdiagnosed with T2D (FIG. 8). The commercially available mouse monoclonalanti-IAPP antibody stained physiological IAPP on pancreatic islets fromthe non-diabetic control patient; see FIG. 8. The antibodies of thepresent invention also gave positive results on diabetic cat pancreasesshowing islet amyloid deposits; see FIG. 9. The same binding propertiesseem to apply to antibodies NI-203.19F2 and NI-203.15C7.

These data demonstrate that NI-203.9A2, NI-203.19H8, NI-203.26C11,NI-203.19F2 and NI-203.15C7 antibodies specifically recognizepathological IAPP fibrils and are in accordance with the biochemicalbinding properties of these antibodies, which show strong bindingspecificity to IAPP fibrils in vitro (FIG. 5).

Example 5: NI-203.9A2, NI-203.19H8 and NI-203.26C11 Antibodies do notCross-React to Pathological Aβ Amyloid in the Brain of Patient Diagnosedwith Alzheimer's Disease

Paraffin-embedded brain sections of a patient diagnosed with Alzheimer'sdisease was used to assess cross-reactivity of the exemplary NI-203.9A2,NI-203.19H8 and NI-203.26C11 antibodies. After formic acid pretreatment,sections were incubated with human NI-203.9A2, NI-203.19H8 andNI-203.26C11 antibodies (50 nM) or mouse monoclonal anti-β amyloidantibody 6E10 (1:2000; Covance, Allschwill, Switzerland), followed byincubation with biotinylated donkey anti-human secondary antibody(1:500; Jackson ImmunoResearch, Newmarket, UK) or biotinylated goatanti-mouse secondary antibody (1:500; Jackson ImmunoResearch, Newmarket,UK). Antibody signal was amplified with the Vectastain ABC-AP kit(Vector Laboratories, USA) and detected with diaminobenzidine substrate(Thermo Fisher Scientific, USA).

NI-203.9A2, NI-203.19H8 and NI-203.26C11 antibodies did not recognizepathological AP amyloid in Alzheimer's disease human brain, in contrastto the anti-β amyloid specific antibody 6E10 (FIG. 10). The same seemsto apply to antibodies NI-203.19F2 and NI-203.15C7.

These data demonstrate that NI-203.9A2, NI-203.19H8 and NI-203.26C11antibodies are not cross-reactive to pathological AP amyloid.Accordingly, also minimal cross-reactive binding of NI-203.9A2,NI-203.19H8 and NI-203.26C11 antibodies to several protein candidateswith misfolding/aggregation propensities by direct ELISA has beendemonstrated, including the most prominent amyloid-forming proteinsincluding, but not restricted to, alpha-synuclein, superoxide dismutase1 (SOD1), Tau and TAR-binding protein 43 (TDP-43).

Example 6: Quality Control of Mouse Chimeric NI-203.9A2, NI-203.19H8 andNI-203.26C11 Antibodies

To validate the exemplary mouse chimeric NI-203.9A2, NI-203.19H8 andNI-203.26C11 antibodies, direct ELISA assays were performed as describedabove. Chimeric antibodies were compared to corresponding humanantibodies. For the exemplary recombinant chimeric NI-203.9A2,NI-203.19H8 and NI-203.26C11 antibodies, and their corresponding humanantibodies, 96-well microplates (Costar, Corning, USA) were coated withhuman IAPP solution or with BSA (Sigma-Aldrich, Buchs, Switzerland)diluted to a concentration of 10 μg/ml in carbonate ELISA coating buffer(15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.42) and binding efficiency of thechimeric and human antibodies was tested. Binding was determined using adonkey anti-human IgGγ antibody (Jackson immunoResearch, Newmarket, UK)conjugated with HRP, followed by measurement of HRP activity in astandard colorimetric assay. Importantly, the human IAPP solution usedfor ELISA assay contained IAPP fibrils, as shown by electron microscopy;see FIG. 3A. The EC₅₀ values were estimated by a non-linear regressionusing GraphPad Prism (San Diego, USA) software.

NI-203.9A2, NI-203.19H8 and NI-203.26C11 mouse chimeric antibodies bindwith a high affinity to human IAPP fibrils with an EC₅₀ of 18.6 nM, 23.9nM and 11.5 nM, respectively. No binding was observed on BSA. Thebinding affinity of chimeric antibodies was similar to their humancounterparts, with an EC₅₀ of 9.4 nM, 22.9 nM and 6.8 nM for humanNI-203.9A2, NI-203.19H8 and NI-203.26C11 antibodies, respectively; seeFIG. 11.

The exemplary mouse chimeric NI-203.9A2, NI-203.19H8 and NI-203.26C11antibodies were further validated on paraffin-embedded pancreas sectionsof two selected patients diagnosed with diabetes mellitus type 2 (T2D),and showing islet amyloid deposits. After formic acid pretreatment,sections were incubated with chimeric NI-203.9A2, NI-203.19H8 andNI-203.26C11 antibodies (50 nM), followed by incubation withbiotinylated donkey anti-human secondary antibody (1:500; JacksonImmunoResearch, Newmarket, UK). Antibody signal was amplified with theVectastain ABC-AP kit (Vector Laboratories, USA) and detected withdiaminobenzidine substrate (Thermo Fisher Scientific, USA). Uponavidin/biotin blocking (Avidin/Biotin blocking kit, Vector Laboratories,USA), pancreatic islet β-cells were visualized using a polyclonal guineapig anti-insulin antibody (1:5; Dako, USA) coupled to a biotinylateddonkey anti-guinea pig secondary antibody (1:500; JacksonImmunoResearch, Newmarket, UK) and antibody signal was amplified withthe Vectastain ABC-AP kit (Vector Laboratories, USA) and detected withalkaline phosphatase substrate (Vector Laboratories, USA).

NI-203.9A2, NI-203.19H8 and NI-203.26C11 chimeric antibodies showedprominent pancreatic islet staining on amyloid-positive sections fromtwo T2D patients (FIG. 12).

These data demonstrate that chimeric NI-203.9A2, NI-203.19H8 andNI-203.26C11 antibodies specifically recognize pathological IAPP fibrilswith efficiency comparable to their human counterparts (FIG. 12).

Example 7: In Vivo Validation of the Therapeutic Effect of the IAPPand/or proIAPP Antibodies in T2D Animal Models

Lead antibody candidates are validated in two transgenic mice models andin a rat model expressing hIAPP: 1) h-IAPP (hemizygous)/C57BL/6/DBA miceexposed to high fat diet (Hull et al. (2003), Diabetes 52: 372-379); 2)h-IAPP (hemizygous)/A^(vy)/A mice exposed to standard diet (Butler etal. (2003), Diabetes 52: 2304-2314); 3) h-IAPP (homozygous)/CD ratsexposed to standard diet (Butler et al. (2004), Diabetes 53: 1509-1516).Therapeutic efficacy is assessed by determining the beta-cell mass andhIAPP amyloid load in the pancreas as well as plasma levels of hIAPP,and functional tests of glucose metabolism and insulin secretion.

1) Physiological Characteristics

The following physiological characteristics (i) to (vii) of the type IIdiabetes animal models are tested to see whether the application of theantibodies of the present application show preventive and/or therapeuticeffect.

(i) Blood Glucose:

The blood glucose level of the T2D animal models is tested and comparedto non-treated animals and a normal (not T2D model) strain animals.

The “normal strain mouse” herein is not particularly limited and can beany mouse as long as it shows no abnormality in the blood glucose level,urea sugar, insulin secretion and the like. Preferred examples of the“normal strain mouse” include a KOR mouse, NC mouse, and laboratorymouse which is used as a recurrent parent in generating a congenic mouse(e.g., C3H/He mouse, BALB/c mouse, and C57BL/6 mouse).

The “normal strain rat” herein is not particularly limited and can beany rat as long as it shows no abnormality in the blood glucose level,urea sugar, insulin secretion and the like.

As diabetes in the mouse and rat models is preferably induced bytransgenic expression of hIAPP, preferably the same strains are used ascontrols as those which were originally used for the generation of thetransgenic animals.

The expression “having a higher blood glucose level as compared to anormal strain mouse/rat” means that the blood glucose level (glucoseconcentration in the blood) at fasting is higher than that of a normalstrain mouse/rat at fasting. Blood glucose levels of the diabeticanimals of 130 mg/dl or higher, more preferably 140 mg/dl or higher,further preferably 200 mg/dl and further more preferably 300 mg/dl orhigher is classified as higher blood glucose. Further, the term“fasting” as used herein means a condition about 12 hours after thestart of fasting of a mouse/rat.

In the present invention, the “blood glucose level” can be measured by aconventional method known to the person skilled in the art, for example,using a commercial measuring apparatus (e.g., Medisafe Reader; TerumoCo., Ltd.) according to the method described in Example hereinafter.

Exemplary Method of Measurement of Blood Glucose Level

The blood glucose level (blood glucose concentration) of a subjectanimal is measured using a commercial measuring apparatus (MedisafeReader, Terumo Co., Ltd.). Measurement principle of this apparatus willbe explained as follows. The measurement is based on colorimetricanalysis. A measuring chip is prepared, and onto the chip are placedglucose oxidase and peroxidase as catalysts and 4-aminoantipyrine andN-ethyl-N(2-hydroxy-3-sulfopropyl)-m-toluidine as chromogenic agents.When a blood sample absorbed through capillary phenomenon is placed onthis chip and then glucose in the blood is oxidized by glucose oxidase.Then, the chromogenic agents on the chip are oxidized by hydrogenperoxide generated at this moment and peroxidase, which yields ared-purple color. The amount of glucose in the blood is calculated bymeasuring the degree of this color tone.

Here, 4 μl of the whole blood is obtained from a subject animal as ablood sample and measured using a measurement time of 18 seconds.

(ii) Glycosylated Hemoglobin (HbA1c) Concentration:

The Type II diabetes animal models are tested for increased bloodglycosylated hemoglobin concentration and compared to non-treated T2Dmodel animals and normal, non-T2D model animals. Concentrations of 2.5%or higher, 2.6% or higher, further 2.8% or higher, and further 3.0% orhigher are classified as increased.

The “glycosylated hemoglobin concentration” as used herein means theproportion of hemoglobin molecules with glucose attached to them in ared blood cell. The glycosylated hemoglobin concentration can be used asan index to judge appropriateness of therapeutic control for diabetespatients and is known to correlate better with the blood sugar level at1 to 2 months earlier than with that at the present time.

The “glycosylated hemoglobin concentration” can be measured by aconventional method known to the person skilled in the art, for example,using a commercial measuring apparatus (e.g., DCA 2000 System; BayerMedical Ltd.) according to the method described in Example hereinafter.More specifically, for example, when the abovementioned DCA 2000 Systemis used as a measuring apparatus, the amount of total hemoglobin ismeasured by the thiocyan-methemoglobin method and the amount ofglycosylated hemoglobin is measured by the latex coagulation inhibitionreaction.

(iii) Urine Sugar:

Urine sugar of the control animals and of the T2D model animals istested herein.

The term “positive in test for urine sugar” as used herein means thatthe glucose concentration in the urine excreted by the animals is 100mg/dl or higher. The urine glucose concentration can be measured by aconventional method, for example, by the method described in Examplehereinafter using a commercial kit (e.g., Pretest; Wako Pure ChemicalIndustries, Ltd.). Specifically, for example, when the Pretest is usedas a measuring kit, a animal urine sample is first put on a test paperof the Pretest, and after 30 seconds a judgment is made according to thespecified color table in this kit for the classification into fivegrades ranging from − to +4. Urine glucose concentrations estimated fromthe result of the judgment are 100-250 mg/dl for +1, 250-500 mg/dl for+2, 500-2000 mg/dl for +3, and 2000 mg/dl or higher for +4. The judgingresults of +1 and higher are assessed as “positive in test for urinesugar”.

Exemplary Method of Measurement of Urine Sugar

Urine glucose (urine sugar) of a subject animal is measured by methodsknown to the person skilled in the art, e.g., using a commercial kit(Pretest; Wako Pure Chemical Industries, Ltd.). First, an animal urinesample is blotted into a test paper of the abovementioned Pretest, andafter 30 seconds judgment is made according to a color table specifiedfor the classification into five grades ranging from − to +4. Urineglucose concentrations estimated from the results of the judgment are100-250 mg/dl for +1, 250-500 mg/dl for +2, 500-2000 mg/dl for +3, and2000 mg/dl or higher for +4. The judging results of +1 and higher areassessed as “positive in test for urine sugar”.

(iv) Blood Insulin Concentration:

It is tested whether the blood insulin concentration of the Type IIdiabetes animal models is compared to the levels in is equivalent to orhigher than that of a non-treated and that of non-diabetic controlanimals (normal strain animals).

The expression that the blood insulin concentration is “equivalent to orhigher than that of a normal strain animal” means that the blood insulinconcentration at fasting is equivalent to or higher than that of anormal strain animal at fasting, e.g., 90 pg/ml or higher, or 110 pg/mlor higher.

In the present invention, the “blood insulin concentration” can bemeasured by a conventional method, for example, using a Levis insulinassay kit U-type (Shibayagi Co.) according to the method described inExample hereinafter. More specifically, for example, an anti-insulinmonoclonal antibody (mouse) is immobilized onto a plate, insulin in asample is bound thereto, after which a biotin-labeled anti-insulinmonoclonal antibody which recognizes another site of insulin is reactedtherewith, a peroxidase-avidin conjugate is further added thereto tobind to biotin, and finally a chromogenic substance is added to measureinsulin by color development.

Exemplary Method of Measurement of Blood Insulin Concentration

The blood insulin concentration of a subject mouse is measured using amethod known to the person skilled in the art, e.g., incorporated into acommercial kit (Levis insulin assay kit U-type; Shibayagi Co.). Theinsulin concentration is measured in the following manner. Ananti-insulin monoclonal antibody is immobilized onto a plate, insulin ina sample is bound to it, after which a biotin-labeled anti-insulinmonoclonal antibody, which recognizes another portion of insulin, isreacted, a peroxidase-avidin conjugate is further added thereto to bindto biotin, and finally a chromogenic substance is added to measureinsulin by color development. The range of measurement is generally from39 to 2,500 pg/ml for a normal animal (mouse).

(v) Glucose Tolerance:

The treated and non-treated T2D model animals and normal animals aretested for abnormal glucose tolerance.

Whether the glucose tolerance of an animal is normal or abnormal can beconfirmed by a glucose tolerance test. The glucose tolerance test can becarried out according to a conventional procedure known to the personskilled in the art, for example, by intraperitoneally administeringglucose to a fasting (at least 12 hours) animal at 2 mg per gram ofbodyweight and measuring the blood glucose level of the animal atcertain times during the glucose tolerance test (for example, every 15minutes over 240 minutes). When the result shows that the blood glucoselevel shows no tendency to decrease with time as compared with that fora normal mouse, the glucose tolerance is assessed as abnormal. When theresult in the treated animals shows tendency to decrease with time ascompared with that for non-treated animals, the treatment with theantibodies of the present invention is classified as effective.

Exemplary Method of Evaluation of Glucose Tolerance

The glucose tolerance of subject animals is evaluated by the glucosetolerance test.

The glucose tolerance test is carried out by first administering glucoseintraperitoneally to a 12-hour fasting animal at 2 mg per gram ofbodyweight and then measuring the glucose level in the blood (peripheralblood) of the animal, every 15 minutes over 240 minutes. The bloodglucose level is measured by the abovementioned method. When the resultshows that the blood glucose level exhibits no tendency to decrease withtime as compared with that for a normal animal, the glucose tolerance isassessed as abnormal. A faster decrease with time in treated compared tonon-treated T2D model animals is assessed as a sign of efficiency of thetreatment of the present invention.

(vi) Insulin Sensitivity:

The treated and non-treated T2D model animals and normal animals aretested for abnormal insulin sensitivity.

Whether the insulin sensitivity of an animal is normal or abnormal canbe confirmed by an insulin sensitivity test. The insulin sensitivitytest can be carried out according to a conventional procedure known tothe person skilled in the art, for example, by intraperitoneallyadministering insulin to a fasting animal at 0.5-0.85 U per kg ofbodyweight and measuring the blood glucose level of the animal atcertain times during the insulin sensitivity test (for example, every 15minutes over 240 minutes). When the result shows that the blood glucoselevel shows no tendency to decrease with time as compared with that fora normal mouse, the insulin sensitivity is assessed as abnormal. Whenthe result in the treated animals shows tendency to decrease with timeas compared with that for non-treated animals, the treatment with theantibodies of the present invention is classified as effective.

Exemplary Method of Evaluation of Insulin Sensitivity

The insulin sensitivity of subject animals is evaluated by the insulinsensitivity test.

The insulin sensitivity test is carried out by first administeringinsulin intraperitoneally to an 12-hour fasting animal at 0.5-0.85 U perkg of bodyweight and then measuring the glucose level in the blood(peripheral blood) of the animal every 15 minutes over 240 minutes. Theblood glucose level is measured by the abovementioned method. When theresult shows that the blood glucose level exhibits no tendency todecrease with time as compared with that for a normal animal, theinsulin sensitivity is assessed as abnormal. A faster decrease with timein treated compared to non-treated T2D model animals is assessed as asign of efficiency of the treatment of the present invention.

(vii) Others:

Assessment of Polydipsia and Polyuria

The Type II diabetes model animals are tested for showing tendencies ofincreased water drinking and increased urination, after the onset ofdiabetes and during the treatment of the antibodies of the presentinvention. The tendency of increased water drinking can be confirmed,for example, by carefully monitoring the rate of decrease in the watervolume in a water bottle placed in a rearing cage and comparing it withthat for a normal strain animal and with that of non-treated animals.Further, the tendency of increased urination can be confirmed, forexample, by observing the extent of wetting of a floor sheet in therearing cage and comparing it as indicated before.

Judgment of the Presence or Absence of Obese Tendency in Mice

The body weights of treated, non-treated T2D model animals and normalanimals are measured and compared. Decreased weight of treated animalsin comparison to non-treated animals and/or comparable weight of treatedand normal animals is assessed as a sign of efficiency of the treatmentof the present invention.

2) Histopathological Examination

The pancreatic tissue of Type II diabetes animals, of the non-treatedT2D model controls and of normal animals is fixed, embedded in paraffinand stained and analyzed according to the methods of Example 4 foramyloid deposits of amylin on pancreatic islet (Langerhans islet)β-cells. Similarly, β-cell apoptosis and β-cell survival is assessed byimmunostaining using appropriate markers (e.g., TUNEL and cleavedcaspase-3 staining for apoptosis and insulin staining for β-cell area).A decrease of amyloid deposits and/or β-cell apoptosis and/or anincrease in β-cell survival in pancreatic islets in treated animals incomparison with non-treated animals or comparable levels in treatedanimals and in normal animals is assessed as a sign of the efficiency ofthe preventive and/or therapeutic methods of the present inventions.

3) Housing of the Animals

The Type II diabetes animal models are maintained under SPF conditionsand accordingly to what previously described (Hull et al. (2003),Diabetes 52: 372-379; Butler et al. (2003), Diabetes 52: 2304-2314;Butler et al. (2004), Diabetes 53: 1509-1516);. At 6 weeks of age,h-IAPP (hemizygous)/C57BL/6/DBA mice are assigned to a high fat dietshown to promote islet amyloid formation (Hull et al. (2003), Diabetes52: 372-379).

1-37. (canceled)
 38. A polynucleotide encoding a human-derivedrecombinant anti-islet amyloid polypeptide (IAPP) antibody orantigen-binding fragment thereof, wherein the antibody orantigen-binding fragment thereof: (a) is capable of binding human IAPP;(b) does not substantially recognize amyloid-β peptide (Aβ₁₋₄₂); and/or(c) does not substantially recognize physiological IAPP
 39. Thepolynucleotide of claim 38, wherein the antibody or antigen-bindingfragment thereof specifically binds an IAPP epitope that comprises theamino acid sequence KCNTATCATQ (SEQ ID NO: 9).
 40. The polynucleotide ofclaim 39, wherein the antibody or antigen-binding fragment thereofcomprises in its variable region at least one complementaritydetermining region (CDR) as depicted in FIG. 1 and/or one or more CDRsthereof comprising one or more amino acid substitutions.
 41. Thepolynucleotide of claim 40, wherein the antibody or antigen-bindingfragment thereof comprises in its variable region an amino acid sequenceof a V_(H) and/or a V_(L) region as depicted in FIG. 1 or a V_(H) and/ora V_(L) region thereof comprising one or more amino acid substitutions.42. The polynucleotide of claim 41, wherein the antibody orantigen-binding fragment thereof comprises at least one of the followingcomplementarity determining regions (CDRs): (a) a CDR-H1 comprising theamino acid sequence of any one of SEQ ID NO: 76, (b) a CDR-H2 comprisingthe amino acid sequence of any one of SEQ ID NO: 94, (c) a CDR-H3comprising the amino acid sequence of any one of SEQ ID NO: 112, (d) aCDR-L1 comprising the amino acid sequence of any one of SEQ ID NO: 85,(e) a CDR-L2 comprising the amino acid sequence of any one of SEQ ID NO:103, (f) a CDR-L3 comprising the amino acid sequence of any one of SEQID NO: 121, or (g) a CDR as in any one of (a)-(f) comprising one or moreamino acid substitutions.
 43. The polynucleotide of claim 42, whereinthe antibody or antigen-binding fragment thereof comprises a variableregion comprising the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO:30, or a variable region comprising an amino acid sequence being atleast 90% identical to the amino acid sequence of SEQ ID NO: 28 or SEQID NO:
 30. 44. The polynucleotide of claim 38, wherein the antibody orantigen-binding fragment thereof preferentially recognizes IAPPaggregates comprising IAPP oligomers and/or fibrils over physiologicalIAPP.
 45. The polynucleotide of claim 38, wherein the antibody orantigen-binding fragment thereof is a chimeric rodent-human or arodentized antibody or antigen-binding fragment thereof.
 46. Thepolynucleotide of claim 38, wherein the antibody or antigen-bindingfragment thereof competes with an antibody for specific binding to IAPP.47. A polynucleotide encoding a human-derived recombinant anti-IAPPantibody or antigen-binding fragment thereof that competes with ananti-IAPP antibody encoded by the polynucleotide of claim 39 forspecific binding to IAPP.
 48. The polynucleotide of claim 38, whereinthe antibody or antigen-binding fragment thereof is selected from thegroup consisting of a single chain Fv fragment (scFv), an F(ab′)fragment, an F(ab) fragment, and an F(ab′)₂ fragment.
 49. Thepolynucleotide of claim 38, wherein the antibody or antigen-bindingfragment thereof (i) comprises a detectable label selected from thegroup consisting of an enzyme, a radioisotope, a fluorophore, and aheavy metal; or (ii) is attached to a drug.
 50. A vector comprising thepolynucleotide of claim
 38. 51. The vector of claim 50, wherein thevector is an expression vector and the polynucleotide is operably linkedto expression control sequences.
 52. A host cell comprising thepolynucleotide of claim 38 or the vector of claim
 50. 53. A method forproducing an anti-IAPP antibody or antigen-binding fragment thereof,said method comprising: (a) culturing the host cell of claim 52 underconditions allowing for expression of the anti-IAPP antibody orantigen-binding fragment thereof; and (b) isolating said anti-IAPPantibody or antigen-binding fragment thereof from the culture.
 54. Ananti-IAPP antibody or antigen-binding fragment thereof encoded by thepolynucleotide of claim
 38. 55. An anti-IAPP antibody or antigen-bindingfragment thereof comprising a variable region comprising: (a) a CDR-H1comprising the amino acid sequence of SEQ ID NO: 76 or a variantthereof, wherein the variant comprises two or fewer amino acidsubstitutions, (b) a CDR-H2 comprising the amino acid sequence of SEQ IDNO: 94 or a variant thereof, wherein the variant comprises two or feweramino acid substitutions, (c) a CDR-H3 comprising the amino acidsequence of SEQ ID NO: 112 or a variant thereof, wherein the variantcomprises two or fewer amino acid substitutions, (d) a CDR-L1 comprisingthe amino acid sequence of SEQ ID NO: 85 or a variant thereof, whereinthe variant comprises two or fewer amino acid substitutions, (e) aCDR-L2 comprising the amino acid sequence of SEQ ID NO: 103 or a variantthereof, wherein the variant comprises two or fewer amino acidsubstitutions, and (f) a CDR-L3 comprising the amino acid sequence ofSEQ ID NO: 121 or a variant thereof, wherein the variant comprises twoor fewer amino acid substitutions.
 56. The anti-IAPP antibody orantigen-binding fragment thereof of claim 55 comprising a variableregion comprising the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO:30, or a variable region comprising an amino acid sequence at least 90%identical to SEQ ID NO: 28 or SEQ ID NO: 30.